CN111684074A - Methods for Determining Microbial Concentrations - Google Patents

Abstract

The present invention provides a method of preparing a suspension of intact microorganisms from a sample comprising microorganisms and mammalian cells, comprising contacting the sample with a buffer solution having a pH of at least pH 6 and less than pH 9, a detergent and one or more proteases to allow lysis of mammalian cells; filter the mixture through a filter suitable for retaining microorganisms to remove lysed mammalian cells; resuspend the microorganisms retained by the filter in a liquid to provide a suspension containing recovered intact microorganisms; and The following methods determine the concentration of microorganisms in the suspension: i. heating and/or contacting an aliquot of the suspension with alcohol; ii. optionally diluting one of the suspensions before, during and/or after step (i) or multiple aliquots to provide one or more diluted aliquots; iii. contacting at least a portion of the aliquots of step (i) or (ii) with a single fluorescent stain capable of binding DNA; iv. imaging the mixture of step (iii) at the emission wavelength of the fluorescent dye and determining an image analysis value corresponding to the number of objects in the imaged mixture; and v. comparing the image analysis value to a predetermined calibration curve , thereby determining the concentration of microorganisms in the suspension.

Description

Methods for Determining Microbial Concentrations

The present invention generally relates to the detection and characterization of microorganisms in a sample. In particular, the present invention provides methods for recovering microorganisms from a sample comprising both microbial cells and non-microbial cells and rapidly measuring the concentration of intact microorganisms recovered from the sample. Whole microorganisms may be alive.

Traditionally, microbial growth and concentration of microorganisms in a sample are determined by measuring an optical parameter of the sample, such as its turbidity. For example, McFarland standards are used in microbiology as a reference for sample turbidity, so the number of microorganisms (usually bacteria) in a sample will be within a given turbidity range, and such standards can be used in a turbidimeter to determine The concentration of microorganisms in the sample. Alternative techniques, including spectrophotometry, can be used to determine the concentration of microorganisms in a sample. However, although fast and easy to implement, such techniques can only approximate the number of microorganisms in a sample. The relationship between turbidity, or absorbance of specific wavelengths of light, and the concentration of microorganisms in a sample also varies with different microbial species, making it difficult to assess the concentration of microorganisms when the identity of the microorganism in question is unclear. Furthermore, such techniques are only able to measure the total turbidity or absorbance of the sample and thus cannot distinguish intact microorganisms or cells or other debris in the sample. Turbidity measurements of the concentration of microorganisms in a sample also have low sensitivity and require relatively high concentrations of microorganisms in order to be able to measure the concentration of microorganisms in a sample. This prevents low concentrations from being measured in this way, and may require extended incubation steps before measurements can be made.

The number of intact microorganisms in a sample can also be estimated more quantitatively by plating a portion (or diluted portion) of the sample on solid growth medium, incubating the sample, and counting the number of colonies formed. The number of colony forming units (CFU) in the plated samples was considered to correspond to the number of viable microorganisms. The disadvantage of this technique, however, is that it requires a lengthy incubation step to allow sufficient time for microbial growth. Thus, such classical techniques can be used to measure the concentration of microorganisms in a sample at a specific point in time, but have limited use in situations where the concentration of intact microorganisms is rapidly required, such as for testing or assaying microorganisms in a sample, which requires Prior knowledge of the number of microorganisms present in the sample.

It is well known in the field of microbial detection that live (ie, live) cells can also be distinguished from dead cells, and many techniques are available for this purpose. Methods known in the art focus on nucleic acid stains, membrane potential, redox indicators or reporter genes. Typically, these techniques rely on the fact that the membranes of living microorganisms are intact, whereas the membranes of dead microorganisms are ruptured and/or destroyed (Gregori et al. 2001. Appl. Environ. Microbiol. 67, 4662-4670).

One specific technique that allows the separation of dead cells from living cells is live/dead staining. By using membrane-impermeable dyes or stains, only cells with damaged membranes are stained, but not cells with intact membranes. Dyes/stains are therefore used as markers for dead cells, since only those cells with damaged membranes (ie dead cells) are stained with such dyes. In this way, dead cells can be detected and in addition the proportion of total dead cells can be calculated. Further developments in this field have led to the development of techniques using two different stains: the first is cell permeable and can enter both living and dead cells; the second is cell impermeable and can only into dead cells. Thus, live and dead cells can be differentially labeled and thus differentiated. An example of a kit for performing this technique is the LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen), which contains SYTO9 (cell permeable) stain and propidium iodide (PI) (cell impermeable) fluorescent stain agent. By detecting microbial cells at the emission wavelengths of both the first and second stains used in such kits, such techniques can be particularly useful for distinguishing between live and dead microorganisms to determine the presence of live microorganisms in a sample proportion.

A number of different detection techniques can be used to distinguish differentially stained live microbial cells from dead microbial cells in a sample. For example, the number of microbial cells indicated as viable and non-viable in the sample can be directly counted, eg, in the microscope field, and in this way the proportion of viable microorganisms present in the sample can be determined. However, such techniques are labor and time intensive and cannot accurately determine the concentration of viable microorganisms. When used in conjunction with live/dead staining techniques, automated cell counting methods such as flow cytometry can also be used to measure the proportion of viable microorganisms in a sample (Berney et al. 2007. Applied and Environmental Microbiology 73, 3283-3290). However, in flow cytometry methods, complex and highly specialized instruments are required as well as periodic calibrations (eg, individual calibrations before each sample is measured). Therefore, such techniques are generally not suitable for use in robust detection methods, such as are required for routine clinical laboratory use, and automation of such techniques can be difficult. Therefore, there is a need for simple, rapid and robust methods and instruments for measuring the concentration of intact microorganisms in a sample, especially for clinical use.

As described above, microorganisms with intact cell membranes can stain differently from microorganisms with damaged or disrupted cell membranes when appropriate dyes are used. Thus, detection of viable cells by live/dead staining generally includes detection of cells with intact cell membranes, and thus cells with intact cell membranes are considered to represent viable cells for the purpose of measuring the concentration of viable microbial cells in a sample. The correlation between intact and viable cells is good, and detection of intact cells is considered an efficient method to determine the number or concentration of viable cells in a sample.

Our co-pending application PCT/EP2018/077852 relates to a method for determining the concentration of intact microbial cells in a sample using "live/dead" stains and imaging. By combining aspects of this method with a particular gentle way of recovering microbial cells from a sample and pre-treating the recovered cells, the inventors of the present application have developed a method that uses only a single dye or stain to A method for determining the concentration of intact microbial cells recovered from a sample provides an improved simplified technique.

As mentioned above, in many situations in microbiology it is desirable to determine the concentration of microorganisms, particularly intact microorganisms. It may be desirable to allow an assay to provide an appropriate concentration or number of microorganisms for use in characterizing the microorganism, so that the assay can be performed correctly, or indeed to ensure that the sample is suitable for a particular assay. Notably, this can include preparing a standard (or standardized) culture or an inoculum of a culture. This includes in particular the preparation of standardized inoculums for antibiotic susceptibility testing (AST), which require an inoculum of known or predetermined or standard concentration for clinical purposes of detection and identification of microbial infections. However, it may also be desirable to determine the concentration of microorganisms in a sample for other assays, or to provide standard cultures, as discussed in more detail below.

Many processes in biology and medicine require an accurate determination of the number of microorganisms (particularly whole/viable microorganisms) in a sample and the preparation of an inoculum based on that determination. These include, for example, water and food quality control analysis, microbial monitoring in environmental samples, biofilm formation in or on medical devices or in patients, and laboratory microbial studies. In particular, accurately determining the concentration of viable microbial cells in a sample and thereby preparing an inoculum containing the desired concentration of microorganisms can be used to diagnose microbial infections.

Microbial infections are a major category of human and animal diseases with significant clinical and economic impact. Although various classes and types of antimicrobial agents can be used to treat and/or prevent microbial infections, antimicrobial resistance is a large and growing problem in modern medicine. Therefore, in the context of treating microbial infections, obtaining information on the nature of infectious microorganisms and their antimicrobial susceptibility profiles may be desirable and very important in order to ensure effective treatment and also reduce the use of unnecessary or ineffective antibiotics, and Thereby helping to control the spread of antibiotic resistance or, more generally, antimicrobial resistance. This is especially true in serious or life-threatening infections where rapid and effective treatment is critical.

Sepsis, a potentially fatal systemic inflammation caused by severe infection, is the most expensive condition in the United States and a driver of hospital costs, accounting for 5% of total national hospital costs. Mortality in severe sepsis increases by 7% per hour if left untreated, and the growing prevalence of antimicrobial-resistant sepsis-causing strains, especially bacterial strains, makes the right treatment for sepsis Predictions are getting harder and harder. The current gold standard for diagnosing microorganisms that cause sepsis or other infections is based on phenotypic and biochemical identification techniques, which require isolation and cultivation of pure cultures of infectious microorganisms. Microbial identification (ID) and antibiotic susceptibility (AST) testing can take several days to identify infections and determine the susceptibility profile of microorganisms that may be resistant to one or more antibiotics. The AST assay provides a "minimum inhibitory concentration" or "MIC" value for each antimicrobial agent tested on a microorganism, and thus can provide information about which antimicrobial agents are likely to be effective against the microorganism. The faster such information can be provided the better, so fast AST methods are desirable and are being developed.

In general, the results obtained from AST determinations should be comparable between different methods and/or between different clinical laboratories. To this end, it is customary to conduct AST tests using prescribed and recognized conditions. This may involve the use of a defined medium (eg Muller-Hinton (MH) medium) and culture conditions. In particular, it is customary to also use a normalized microbial titer (ie a normalized (or standard) number or amount (eg concentration) of microbial cells) to establish cultures performed in an AST test (ie to monitor growth) such that the culture The number or amount of bacteria in the set value. For example, the McFarland standard is routinely used as a reference for adjusting the turbidity of microbial suspensions, especially bacterial suspensions, to bring the number of microorganisms in a culture formulation used in establishing cultures within a given range for AST testing standardization. The McFarland standard is set based on the turbidity of the reference suspension, and the concentration (or bacterial count) of the microbial suspension is adjusted to match the turbidity of the selected McFarland standard.

Conventionally (eg, as described in EUCAST Standard Methods for Determining the MIC of Antimicrobial Agents (European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID), Clinical Microbiology and Infection, Vol. 9 ( 8):ix–xv, 2003)), microbial cells for AST (eg from clinical sample cultures) are plated and incubated to obtain isolated colonies. Colonies can then be collected and used to prepare microbial cell suspensions for use as inoculum for AST assays. Typically, and as described in the guidelines above, the concentration of microorganisms in the suspension so prepared is set to a standard and predetermined level, eg, 0.5 McFarland units, to allow standard concentrations of microorganisms to be used for AST determinations. The turbidity of the microbial suspension can be adjusted to 0.5 McFarland units prior to use. Alternatively, isolated single colonies can be used to inoculate the medium, which can be grown to provide the inoculum. Cultures can be allowed to grow to the desired standard (0.5 McFarland units) and/or adjusted to this standard if necessary prior to use as an inoculum. Therefore, microbial cultures are typically grown until growth reaches a turbidity equal to or greater than the 0.5 McFarland standard prior to normalizing bacterial concentrations prior to AST. If desired, cultures can be adjusted to provide cultures with a turbidity equal to the 0.5 McFarland standard. This can then be used as an inoculum for establishing an AST assay. The inoculum obtained at this point (ie about 0.5 McFarland units of culture or suspension) was diluted in broth to obtain the desired normalized final cell number concentration for AST culture. For reference, a 0.5 McFarland unit of microbial culture/suspension contains a microbial concentration of about ¹ x 108 CFU/ml. When establishing AST cultures, such microbial cultures/suspensions are typically diluted ~200-fold in broth, ie each AST culture condition will typically contain a starting microbial concentration of about ^5x105 CFU/ml.

For certain microbial infections, such as sepsis, blood samples are typically collected in blood culture bottles and microbial cultures (ie, clinical sample cultures) are allowed to grow until a positive culture result is obtained in a culture monitoring system. In automated culture detection systems, such as the ^Bactec or Bact/Alert systems, the bacterial concentration required to indicate a positive is between 108 and ¹⁰⁹ CFU/ml, corresponding to 0.5 to 3.5 McFarland units (if in saline solution) Measurement). The lowest McFarland value that is easily detectable (measured by eye or turbidimetry) is about 0.5 McFarland units.

Typically once a positive culture result is obtained, such clinical sample cultures can be used for ID testing and AST determination. For AST testing, further cultures are typically prepared from clinical sample cultures (eg, positive cultures) for use as or for preparing inoculum for AST test cultures, and such before they are used to inoculate AST test cultures The inoculum was normalized to a preset microbial concentration or McFarland value (usually 0.5 McFarland units). Therefore, an inoculum of AST is typically prepared using or from 0.5 McFarland units of culture or microbial suspension. As described above, this is typically accomplished in the methods of the art by selecting colonies obtained by plating clinical sample cultures or microorganisms isolated therefrom.

Techniques that require comparison to McFarland standards to determine the concentration of microorganisms in a sample can only provide an approximation of the concentration and cannot provide specific information about the concentration of viable microbial cells in the sample. Furthermore, such techniques rely on relatively high (eg, 0.5 McFarland units) microbial concentrations in the sample in order to measure the concentration.

There is therefore a particular need to improve the speed and sensitivity of determining the concentration of microorganisms in a sample, especially where AST assays are established. In particular, there is a need for robust and simple methods that allow for rapid, accurate and sensitive determination of microbial concentrations without the need for complex instrumentation, such as methods including flow cytometry. The present invention addresses this need by providing an improved method for determining microbial concentration that can be used to prepare a microbial inoculum, and further providing an improved workflow for performing AST that allows accurate and rapid Determine the concentration of microorganisms in the microbial suspension, more notably the concentration of intact microorganisms in the microbial suspension. In particular, the concentration determination method of the present invention is valuable in enabling rapid AST assays. Thus, the present invention provides a rapid, accurate and precise method for determining the concentration of microorganisms, more notably intact microorganisms, in microbial preparations. As mentioned above, the concentration of intact microorganisms can be used as a reliable indicator of viable microorganisms.

In particular, the method of the present invention is based on recovering microbial cells from a sample in a manner that is particularly effective for separating microbial cells from non-microbial cells, especially mammalian cells, by lysing the non-microbial cells while leaving the microorganisms intact (and largely or substantially viable), the recovered microbial suspensions are stained for intact microorganisms, and the suspensions are imaged to determine a value corresponding to the number of objects that are intact microorganisms in the sample, but not for microorganisms The number of viable microorganisms present is counted by direct counting or by nephelometric estimation of the concentration of microorganisms relative to a predetermined standard or by counting cultured colonies. By using a predetermined standard curve, a determined value of the number of objects detected by imaging can be correlated with the concentration of microorganisms present in the suspension. By combining this gentle (maintaining viability) isolation (microbial isolation) technique with the step of pre-treating the recovered microbial cells with alcohol and/or heat to aid or assist the staining process prior to staining, it was unexpectedly discovered that , only a single stain can be used reliably to detect and determine the microbial concentration in recovered microbial suspensions. Without wishing to be bound by theory, we believe that the separation step produces a sufficiently pure preparation of microbial cells from the sample that is largely (eg, substantially or substantially) viable (eg, only a small percentage of non-viable microbial cells), which allows the use of only a single stain. This combined effect with pretreatment is particularly beneficial for the quantification of microorganisms, especially bacteria, that have resistance mechanisms that affect cell permeability and thus the ability of microorganisms to take up and/or retain stains. Without wishing to be bound by theory, it is believed that pretreatment steps can disrupt or disable efflux pumps in microorganisms (a common mechanism of antimicrobial resistance), thereby enhancing resistant microorganisms (particularly or indeed those with strong or any microorganisms that are effective in efflux pumps). In other words, staining of microorganisms, particularly antimicrobial-resistant microorganisms, can be normalized by using the pretreatment steps described herein. This is important because errors in the AST for drug-resistant bacteria are especially detrimental to patients from whom the bacteria have been isolated, as this increases the risk of incorrect treatment.

Accordingly, in a first aspect, the present invention provides a method of preparing a suspension of intact microorganisms from a sample comprising microorganisms and mammalian cells, the method comprising:

a. Provide samples containing microorganisms and mammalian cells;

b. contacting the sample with a buffer solution, a detergent and one or more proteases, wherein the pH of the buffer solution is at least pH 6 and less than pH 9 to allow lysis of mammalian cells present in the sample;

c. filtering the mixture obtained in step (b) through a filter suitable for retaining intact microorganisms, wherein said filtering removes lysed mammalian cells from said mixture;

d. recovering the microorganisms retained by the filter in step (c), wherein the recovering comprises resuspending the microorganisms in a liquid to provide a suspension comprising the recovered intact microorganisms; and

e. Determine the concentration of microorganisms in the suspension, wherein the concentration of microorganisms is determined by a method including:

i. contacting an aliquot of the suspension with alcohol and/or heating an aliquot of the suspension;

ii. optionally diluting one or more aliquots of the suspension to provide one or more diluted aliquots at one or more dilution values, wherein the dilution is prior to step (i) , during and/or after;

iii. contacting at least a portion of the aliquot of step (e)(i) or (e)(ii) with a single fluorescent stain capable of binding DNA to provide a suspension-stain mixture, wherein the stain has emission wavelength;

iv. imaging the suspension-stain mixture of step (e)(iii) at the emission wavelength of the fluorescent stain, and determining an image analysis value corresponding to the number of objects in the imaged mixture corresponding to microorganisms; and

v. Compare the image analysis values obtained in step (e)(iv) for the aliquot of step (e)(iii) with a predetermined calibration curve to determine the microbial population in the suspension concentration.

It will be understood from the above that step (b) is a step of selectively lysing non-microbial cells present in the sample, leaving the microbial cells in the sample intact (or more particularly substantially intact). Thus, in step (b), the detergent is used in an amount or concentration effective to lyse (or to lyse or capable of lysing) non-microbial cells but not effective to lyse (or incapable of lysing or incapable of lysing) microbial cells.

Steps (e)(i) of pretreating the microorganisms in suspension with alcohol and/or heat, as described above, are used to facilitate subsequent staining. Without wishing to be bound by theory, this may be due, at least in part, to the role of pretreatment in permeabilizing the cell walls and/or membranes of microorganisms, or otherwise affecting conformational changes in the microbial structure to facilitate entry of stains and /or retention, and/or inactivation of microorganisms, eg, so that stains are not removed from microbial cells by efflux pumps. As mentioned above, it is believed that the inactivation of efflux pumps in microorganisms (when present) contributes significantly to the beneficial effects of this method. Thus, alternatively, pretreatment in step (e)(i) can be used to normalize staining.

Although alcohol and/or heat provide an effective such pretreatment, this can also be achieved by other means, such as the use of detergents, for example at concentrations capable of producing a similar (eg permeabilization and/or inactivation) effect on microorganisms or quantity.

Accordingly, in another aspect, the present invention provides a method of preparing a suspension of intact microorganisms from a sample comprising microorganisms and mammalian cells, the method comprising:

a. Provide samples containing microorganisms and mammalian cells;

b. contacting the sample with a buffer solution, a detergent and one or more proteases, wherein the pH of the buffer solution is at least pH 6 and less than pH 9 to allow lysis of mammalian cells present in the sample ;

c. filtering the mixture obtained in step (b) through a filter suitable for retaining microorganisms, wherein said filtering removes lysed mammalian cells from said mixture;

d. recovering the microorganisms retained by the filter in step (c), wherein the recovering comprises resuspending the microorganisms in a liquid to provide a suspension comprising the recovered intact microorganisms; and

e. Determine the concentration of microorganisms in the suspension, wherein the concentration of microorganisms is determined by a method including:

i. contacting an aliquot of the suspension with a detergent;

ii. optionally diluting one or more aliquots of the suspension to provide one or more diluted aliquots at one or more dilution values, wherein the dilution is prior to step (i) , during and/or after;

iii. contacting at least a portion of the aliquot of step (e)(i) or (e)(ii) with a single fluorescent stain capable of binding DNA to provide a suspension-stain mixture, wherein the stain has emission wavelength;

iv. imaging the suspension-stain mixture of step (e)(iii) at the emission wavelength of the fluorescent stain, and determining an image analysis value corresponding to the number of objects in the imaged mixture corresponding to microorganisms; and

v. Compare the image analysis values obtained in step (e)(iv) for the aliquot of step (e)(iii) with a predetermined calibration curve to determine the microbial population in the suspension concentration.

In particular, in step (b), the detergent is effective (or used to) achieve (or is able to achieve) selective lysis of non-microbial cells (ie, lysis of non-microbial cells in the sample, but not lysis of microbial cells), while In step (e)(i), the detergent is effective (or used to) promote (or can promote), for example, enhance or improve or allow or normalize microbial cells, especially antimicrobial resistant microbial cells or have strong Staining of microorganisms in efflux pumps. Therefore, although the same or different detergents may be used in steps (b) and (e)(i), when the detergents are the same, in step (e)(i) compared to step (b) Used in different (higher) amounts.

In the above methods, the fluorescent stain may be cell permeable or cell impermeable, but in preferred embodiments it is cell permeable.

While pretreatment steps can affect the permeability of the cell membrane and/or cell wall of microorganisms, and thus the integrity of the cell wall and/or membrane, we have found that this does not diminish the ability to detect and image microorganisms for enumeration Objects corresponding to microorganisms. Thus, objects corresponding to microorganisms can be identified and imaged. Although the integrity of the cell walls and/or membranes may be compromised to some extent during the pretreatment step, the imaged objects can be identified as corresponding to the microorganisms recovered intact in step (d). Thus, the image analysis value obtained in step (e)(iv) can be considered as a value corresponding to (or representative of) the number of objects in the imaged mixture of recovered intact microorganisms. Thus, in step (e)(v), the comparison step allows to determine the concentration of intact microorganisms in the suspension (ie in the suspension prepared in step (d)).

Comparison of the image analysis value of the number of objects detected in step (e)(v) with a predetermined calibration curve enables a more accurate measurement of the number of microorganisms (or more particularly intact microorganisms) in the suspension to be obtained. A variety of factors can affect staining and/or identify intact cells by staining methods. For example, in the case of live/dead staining, it has been reported in some cases that while a portion of a microbial cell indicated as "live" in a live/dead staining assay may contain an intact cell membrane, it may in fact be metabolic Inactivated or otherwise unculturable (Trevors 2012. J Microbiol Meth 90, 25-8). Furthermore, during rapid exponential growth in a nutrient-rich environment, the membrane integrity of living microbial cells may be reduced, allowing a second fluorescent stain to enter the cells (Shi et al. 2007. Cytom Part A71A, 592-298). Thus, such cells will emit light at the second emission wavelength, and the fluorescence of such cells at the first emission wavelength may be reduced due to the ability of the second fluorescent stain to quench the fluorescence of the first fluorescent stain. Additionally, issues such as bleaching and higher than expected uptake of the first (cell-permeable) fluorescent stain may affect the accuracy of such methods (Stiefel et al. 2015. BMC Microbiology 15:36). The methods disclosed herein allow consideration of these factors that may adversely affect the determination of intact cell concentration in a sample (ie, "affect" any such determination), resulting in a more accurate measurement of microbial viability. Thus, concentrations determined for intact microbial cells can be used to represent, indicate, or correspond to or approximate the concentration of viable microbial cells. In particular, by comparing the image analysis value of the number of objects imaged in step (e)(iv) with a predetermined calibration curve, when trying to calculate the concentration of intact, especially viable microorganisms present in the suspension, one can take into account Factors such as the above-mentioned incorrect staining of live and non-viable microbial cells allow a more accurate determination of the concentration of intact or live microorganisms in the suspension to be performed.

The present invention provides a rapid and sensitive method for determining the concentration of microorganisms in a suspension prepared from a sample (or, alternatively, a sample expressed as recovered microorganisms, a "recovered microorganism sample"). This may have many uses, and in many cases it may advantageously provide a robust and simple method for determining microbial concentrations in recovered microbial samples. In addition to accurately determining the absolute concentration of microorganisms, the method can also have utility in giving an indication of the microbial load in a sample, and thus can be used in any method or situation where it is desired to know or estimate or want to know how many microbial cells are present. Therefore, the situations in which this method can be used are not limited. Indeed, given the lower limitations of detection by this method, this method can be used to determine whether a sample contains microorganisms. Accordingly, in one aspect, the present invention provides a method for determining the presence of microorganisms in a sample, the method comprising performing steps (a) to (e) of any of the above methods disclosed herein, and determining whether the sample is present or not microorganism.

The method of the present invention can be used in situations where it may be desirable to assess or determine the concentration of microorganisms in different samples or suspensions. The sample contains both microorganisms and mammalian cells, and is therefore preferably from a mammal. The sample may in particular be a clinical sample or a veterinary sample, as discussed further below. This method can be used to determine if a sufficient or appropriate concentration of cells is recovered from a sample to enable further testing. This will be described further below in the context of the AST assay, but this method can be used as a preliminary step before any subsequent steps of analyzing the microorganisms in the sample. For example, the method can be used to determine or assess the concentration of intact (or viable) microorganisms in a sample prior to performing mass spectrometry tests and/or nucleic acid-based tests and/or any other assessment of microorganisms (eg, growth-based studies).

Once the concentration of intact (or live) microorganisms in the recovered suspension of microorganisms is determined, this information can be advantageously used to accurately prepare an inoculum containing a known or desired number or concentration of microorganisms.

Accordingly, in another aspect, the present invention provides a method of preparing a microbial inoculum (or alternatively expressed, an inoculum for preparing a microbial culture), the method comprising recovering and determining the amount in suspension using the methods defined herein The concentration of microorganisms is then adjusted to the desired concentration of the microbial cells in at least an aliquot or portion of the suspension, thereby providing an inoculum comprising the desired concentration of microorganisms.

Once the concentration of microorganisms in a suspension containing microorganisms recovered from the sample has been determined, the present invention also provides methods for characterizing the microorganisms in the sample. Thus, the recovery and concentration determination methods of the present invention can be used in conjunction with assays for characterizing microorganisms. In particular, this may be an assay requiring a known or predetermined concentration or number of microorganisms.

Accordingly, in another aspect, the present invention provides a method for characterizing microorganisms in a sample, the method comprising:

(i) providing samples comprising microorganisms and mammalian cells;

(ii) subjecting said sample to steps (b) to (d) as defined above to produce a suspension of intact (eg live) microorganisms;

(iii) performing step (e) as defined above to determine the concentration of microbial cells in the suspension;

(iv) if necessary, adjusting the concentration of microbial cells in the suspension to a desired or predetermined concentration; and

(v) Characterization of the microorganisms in the suspension (and thus the sample).

Thus, the present invention allows determination of the concentration of microorganisms in a preparation (suspension) of recovered microorganisms prior to performing an assay, in particular an assay requiring a specific concentration or number of microorganisms to characterize said microorganisms. This thus allows to determine whether a sample, or more particularly a suspension prepared therefrom, is suitable for a given assay, and if not, to adjust the concentration of microorganisms appropriately.

While concentration adjustment steps in any of the methods set forth herein can be beneficially informed by the determined concentration of microorganisms in suspension, all steps that do not require concentration adjustment are performed after concentration determinations have been completed (eg, in the methods described above). after step (iii)). In one embodiment, the adjustment may be performed after the concentration has been determined, eg, one or more dilution steps may be performed after the concentration has been determined. However, in other embodiments, the initial (ie, preliminary) conditioning step may be performed before the concentration determination step is completed, or independently, eg, when or before the concentration determination step is performed. For example, a preliminary dilution step of the suspension or a portion thereof may be performed before the concentration determination (this is separate from the optional dilution step of the aliquot in step (e)(ii) of the concentration determination method). In such embodiments, one or more additional dilution steps may then be performed after the concentration has been determined to achieve the desired concentration (ie, the dilution resulting from such an initial (preliminary) dilution may be further diluted). Such additional dilutions are informed by the determined concentrations. It will be understood in this regard that such an initial (or preliminary) dilution step (which may be considered a "blind" dilution step) will be performed on a portion of the suspension different from the suspension for which the concentration determination is made, etc. subsample. Thus, the remainder of the suspension (ie the suspension remaining after an aliquot for concentration determination has been taken) can be conditioned (eg diluted) in a preliminary conditioning step, or the suspension (ie the remaining suspension ) for the preliminary conditioning steps in individual sections or aliquots. This can speed up the overall approach.

In another aspect, the present invention provides a method for determining the antimicrobial susceptibility of microorganisms in a sample, the method comprising:

(i) providing a sample comprising live microorganisms and mammalian cells;

(ii) subjecting said sample to steps (b) to (d) as defined above to produce a suspension of said viable microorganisms;

(iii) performing step (e) as defined above to determine the concentration of microbial cells in said suspension;

(iv) using the suspension of step (ii) to inoculate a series of test microbial cultures for antibiotic susceptibility testing (AST), wherein the series of test microbial cultures comprises at least two different growth conditions, wherein the The different growth conditions include one or more different antimicrobial agents, and each antimicrobial agent is tested at two or more different concentrations; and

(v) assessing the extent of microbial growth under each growth condition;

wherein, if desired, the concentration of microbial cells in the suspension or the test microbial culture is adjusted to a desired or predetermined concentration; and

Wherein the degree of microbial growth under each growth condition is used to determine at least one value indicative of the susceptibility of the microorganisms in the sample to at least one antimicrobial agent.

In one embodiment, at least one MIC and/or SIR value can be determined to determine the antimicrobial susceptibility of the microorganism in the sample.

SIR is well known and understood in the art to mean sensitive, intermediate or resistant. Although SIR has a greater process scope than MIC, SIR is used clinically in many settings.

Accordingly, the present invention provides a more accurate method for performing AST determinations, as it allows for a more accurate determination than determining the turbidity of a sample (eg by a simple comparison of the sample turbidity to the McFarland standard turbidity) the concentration of microorganisms. This method is also simpler than using two "live/dead" stains because only a single stain is used. Another advantage of the method is that the concentration of drug-resistant microorganisms can be determined, and in one embodiment, the microorganisms are drug-resistant microorganisms, especially drug-resistant bacteria. As noted above, mechanisms of resistance to antimicrobial agents in microorganisms, particularly bacteria, may include more resistant cell walls and/or membranes, and/or efflux pumps that remove antimicrobial agents from microbial cells. Such mechanisms can also be used to prevent microbial uptake and/or retention of stains. It is believed that the methods of the present invention, including in particular a pretreatment step, can facilitate (or enhance) the staining process (especially antimicrobial resistant microbial cells) to allow detection or measurement of such resistant microorganisms. In other words, the method of the present invention, in particular the pretreatment step, can normalize the staining of microorganisms. We compared resistant and non-resistant bacteria and different types of bacteria with or without pretreatment, and observed enhanced similarity of staining (ie, normalization of staining) between different bacteria. Thus, the staining and methods of the present invention can be performed without knowing the identity of the microorganisms.

Thus, the concentration determination step of the method disclosed above can be used to determine the concentration of drug-resistant microorganisms, particularly drug-resistant bacteria, and may further have more general applicability for determining the concentration of microorganisms in any suspension or preparation of microorganisms .

Accordingly, also disclosed herein is a method for determining the concentration of intact microorganisms in a microbial suspension, the method comprising:

(i) providing a suspension containing microorganisms;

(ii) contacting an aliquot of said suspension with alcohol and/or detergent and/or heating an aliquot of said suspension;

(iii) optionally diluting one or more aliquots of the suspension to provide one or more diluted aliquots at one or more dilution values, wherein the dilution is performed in step (i) before, during and/or after;

(iv) contacting at least a portion of the aliquot of step (ii) or (iii) with a single fluorescent stain capable of binding DNA to provide a suspension-stain mixture, wherein the stain has an emission wavelength;

(v) imaging the suspension-stain mixture of step (iv) at the emission wavelength of the fluorescent stain, and determining an image analysis value corresponding to the number of objects in the imaged mixture corresponding to microorganisms; and

(vi) Comparing the image analysis values obtained in step (v) for the aliquot of step (iv) with a predetermined calibration curve to determine the concentration of microorganisms in the suspension.

With regard to the above method, the image analysis value determined in step (v) may be the number of objects in the imaging mixture for the imaged mixture corresponding to intact microorganisms, and the concentration of intact microorganisms in the suspension may thus be determined in step (vi).

Furthermore, as mentioned above, in one embodiment of the method, the microorganisms may be drug-resistant microorganisms, more particularly drug-resistant bacteria. Still further, such a method can be used in the context of an AST assay and thus, similar to the method described above, can be used as part of a method of determining the antimicrobial susceptibility of microorganisms in a sample.

As mentioned above, standard AST assays according to EUCAST or CLSI guidelines typically require a period of time for the microorganisms to grow sufficiently for the next step to establish an AST assay. For example, in the protocol outlined above, an incubation time is required to allow the concentration of microorganisms in the clinical sample culture to increase to the point where the clinical sample culture is considered "positive" (ie, at least 0.5 McFarland units are reached). A further incubation step is required after the plating of the clinical sample culture to allow growth of individual colonies and, optionally, a further incubation step is required to allow the microbial suspension prepared as outlined above to reach 0.5 McFarland units before the AST assay can be performed .

Furthermore, in the protocol outlined above for preparing an AST assay, typically only one or a small number of colonies (relative to the total number of microorganisms present in the clinical sample culture) are used to prepare the inoculum that is ultimately used to establish the AST assay. Thus, such protocols rely on the use of one or more colonies representing the infection-causing microorganism. If this is not the case, the results of the AST assay may not truly reflect the antimicrobial susceptibility of the infection-causing microorganism, and therefore any clinical intervention based on such results may not adequately treat the infection.

More broadly, the present invention provides qualitative or quantitative assays for the rapid and accurate determination of the concentration of intact microorganisms in suspensions recovered from samples to allow the use of suitable concentrations or numbers of microbial cells to characterize the microorganisms. . In other words, the concentration of intact microorganisms in the recovered suspension can be determined prior to any desired method of characterization of the microorganisms in order to provide a suitable concentration or number of microbial cells for the characterization method. Thus, this allows the use of any such assay to characterize microorganisms.

Assays for which it may be particularly advantageous to determine the concentration of intact microorganisms in microbial suspensions recovered from samples include, for example, mass spectrometry (including MALDI-TOF, ESI-MS and CyTOF), Raman spectroscopy, nucleic acid-based Tests (including PCR, Rolling Circle Amplification (RCA), Ligase Chain Reaction (LCR) and Nucleic Acid Sequence Based Amplification (NASBA), which may be useful in identifying microorganisms and/or markers of antimicrobial resistance therein Particularly useful. As described in more detail elsewhere herein, it can be particularly beneficial to determine the concentration of intact microorganisms in a suspension prepared from a sample prior to performing an AST assay.

As used herein, the terms "microbial cell" and "microorganism" are interchangeable and are considered to have equivalent meanings, ie, microscopic organisms. The term is used broadly herein to include all classes of microorganisms, unicellular or not, and includes bacteria (including mycobacteria), archaea, fungi, protists (including protozoa), and algae, as hereinafter Discussed in more detail. The identity of the microorganism can be known or unknown when the method is performed. Furthermore, a sample may contain one type or species of microorganisms, or more than one type or species, ie the sample may contain a single type of microorganism or may contain a mixture of microorganisms.

Furthermore, references to "cell-permeable" and "cell-impermeable" stains are directed to microbial cells. In other words, the permeability of the dye used in the method of the present invention is the permeability of the dye to the microorganism.

In the context of the present invention, the term "viable" refers to microorganisms capable of growth and/or reproduction. The concentration of viable microorganisms in a sample can be indirectly determined by staining differences to determine the concentration of intact microorganisms in the sample. Therefore, the concentration of viable microorganisms is derived from the concentration of intact cells in the sample. The methods of the present invention provide an accurate and rapid method for determining the concentration of intact microorganisms in a sample. When a sample comprising viable microorganisms is used in step (a), the determination of the concentration of intact microorganisms according to the present invention reflects or provides an indication of the concentration of viable microorganisms.

The term "intact" in the context of microorganisms present in the sample and recovered from the sample and present in the prepared suspension means that the integrity of the microorganism is not substantially altered. Such "intact" microorganisms typically have unruptured cell membranes, ie, cell membranes that are semi-permeable and maintain membrane potential (ie, have a protein gradient). However, as mentioned above, pretreatment with alcohol or heat (or detergent) may have permeabilization effects, and therefore after pretreatment, microorganisms may not be intact in the strict sense of the above definition. Nevertheless, the microorganisms thus pretreated represent the intact microorganisms present in the suspension, and thus the determination of their concentration in the pretreated aliquot (of step (e)(i)) is indicative of the intactness in the suspension the concentration of microorganisms. Furthermore, the permeabilization of the pretreatment can be relatively mild, if at all, and not sufficient to completely destroy the microbial cells.

As mentioned above, the present invention provides methods for preparing suspensions of whole microorganisms. The term "suspension" as used herein has its ordinary meaning as known in the art, ie, a mixture comprising particles. In the present case, "particles" are microorganisms, and a microbial suspension in the methods herein is simply a preparation containing the microorganisms in a liquid. In detail, suspensions were prepared from samples containing microorganisms and mammalian cells.

A range of samples containing a range of possible microorganisms can be analysed in the method of the invention. As mentioned above, the sample contains microorganisms and mammalian cells. However, it is to be understood that it is not possible to determine whether a sample of interest contains microorganisms prior to carrying out the method of the present invention. The sample is preferably isolated from a mammal, but this is not required, and the sample can come from elsewhere, eg it can be an environmental sample. The sample may be known to contain mammalian cells (eg, if it is derived from a mammal), or only suspected to contain mammalian cells, or it may be thought that the sample may contain mammalian cells. Thus, as defined herein, a "sample comprising microorganisms and mammalian cells" may be a sample suspected of containing microorganisms and mammalian cells.

The microorganism can be any microorganism (e.g., any bacterial or fungal microorganism or protozoa, especially any pathogenic microorganism or any microorganism that causes an infection in the body, and thus, the methods of the present invention are particularly useful in detecting or diagnostically testing a subject. The concentration of microorganisms is determined in the case of microbial infection in or on any part of the body (i.e. any microbial infection). In general, the present invention relates to the analysis of samples containing (or suspected of containing) clinically relevant microorganisms, but microorganisms may be Pathogenic or non-pathogenic.

As used herein, the term microorganism includes any organism that may belong to the category of "microorganisms". Although not required, the microorganism can be unicellular, or can have a unicellular life stage. Microorganisms can be prokaryotic or eukaryotic, and generally include bacteria, archaea, fungi, algae, and protists, including protozoa in particular. Of particular interest are bacteria, which can be Gram-positive or Gram-negative, or Gram-indeterminate or Gram-non-responsive, and fungi, such as yeast.

Bacteria can be aerobic or anaerobic. The bacteria may be or may include mycobacteria.

Bacterial genera of particular clinical relevance include Staphylococcus (including coagulase-negative Staphylococcus), Clostridium, Escherichia, Salmonella, Pseudomonas Pseudomonas, Propionibacterium, Bacillus, Lactobacillus, Legionella, Mycobacterium, Micrococcus, Fusiform Fusobacterium, Moraxella, Proteus, Escherichia, Klebsiella, Acinetobacter, Burkhall Burkholderia, Enterococcus, Enterobacter, Citrobacter, Haemophilus, Neisseria, Serratia ( Serratia), Streptococcus (including α-hemolytic Streptococcus and β-hemolytic Streptococcus), Bacteriodes, Yersinia, and Stenotrophomonas (Stenotrophomonas), and indeed any other small or coliform flora. Beta-hemolytic streptococci include Group A, Group B, Group C, Group D, Group E, Group F, Group G and Group H Streptococcus.

Non-limiting examples of clinically relevant Gram-positive bacteria include Staphylococcus aureus (including methicillin-resistant Staphylococcus aureus, MRSA), Staphylococcus haemolyticus, Staphylococcus epidermidis), Staphylococcus saprophyticus, Staphylococcus lugdunensis, Staphylococcus schleiferi, Staphylococcus caprae, Streptococcus salivarius, Streptococcus agalactiae agalactiae, Streptococcus anginosus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus sanguinis, Streptococcus mitis, Streptococcus equi (Streptococcus equinus), Streptococcus bovis, Clostridium perfringens, Clostridium sordellii, Clostridium novyi, Clostridium botulinum , Clostridium tetani, Enterococcus faecalis and Enterococcus faecium. Non-limiting examples of clinically relevant Gram-negative bacteria include Escherichia coli, Salmonella bongori, Salmonella enterica, Citrobacter koseri, Citrobacter freundii, Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas aeruginosa, Haemophilus influenzae, Neisseria meningitidis, Enterobacter cloacae, Enterobacter aerogenes, Serratia marcescens, Stenotrophomonas maltophilia, Morocco Morganella morganii, Bacteriodes fragilis, Acinetobacter baumannii and Proteus mirabilis.

Non-limiting examples of clinically relevant fungi include yeast, especially fungi of the genus Candida, as well as Aspergillus, Fusarium, Penicilium, Pneumocystis ), Cryptococcus, Coccidiodes, Malassezia, Trichosporon, Acremonium, Rhizopus, Trichosporon Fungi of the genera Mucor and Absidia. Of particular interest are the genera Candida and Aspergillus, including Aspergillus fumigatus, Candida albicans, Candida tropicalis, Candida glabrata, Candida dubliensis, Candida parapsilosis and Candida krusei.

Non-limiting examples of clinically relevant protozoa include Entamoebahistolytica, Giardia lamblia, Trypanosoma brucei, Besnoitia besnoiti, Besnoitia bennetti, Besnoitia tarandi, Isospora canis, Eimeria tenella, Cryptosporidium parvum ( Cryptosporidium parvum), Hammondia heydorni, Toxoplasmosa gondii, Neosporacaninum, Hepatozoon canis, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi.

The term "mammalian cell" encompasses any cell of mammalian origin. The cells can be derived from any mammal, particularly humans (ie they can be human cells). Cells may be derived from livestock, such as farm animals such as horses, donkeys, sheep, pigs, goats or cattle, or animals commonly kept as pets such as cats, dogs, mice, rats, rabbits, guinea pigs or chinchillas. A cell can be any type of cell. In particular embodiments, the cells are blood cells, such as red blood cells (erythrocytes) or white blood cells (leukocytes), such as neutrophils, monocytes, or lymphocytes. Platelets are considered herein to be blood cells.

As mentioned above, the sample comprising microorganisms and mammalian cells may be any such sample, but is or is derived from, in particular, a clinical sample or a veterinary sample. A clinical sample is any sample obtained from a human. Thus, it may be any sample of bodily tissue, cells or bodily fluids, or any sample derived from the body, such as swabs, washes, aspirates or washes, and the like. Suitable clinical samples include, but are not limited to, the following samples: blood, serum or plasma, blood fractions, synovial fluid, urine, semen, saliva, feces, cerebrospinal fluid, gastric contents, vaginal secretions, mucus, tissue biopsies Samples, tissue homogenates, bone marrow aspirates, bone homogenates, sputum, respiratory samples, wound exudates, swabs and swab washes such as nasopharyngeal swabs, other body fluids, etc. In a preferred embodiment, the clinical sample is blood or a blood-derived sample, such as serum or plasma or blood fraction. A veterinary sample is an equivalent sample derived from a non-human animal, in this case a non-human mammal. As discussed further below, the sample can also be a clinical sample or a culture of a veterinary sample, such as a blood culture.

The nature of a clinical or veterinary sample can be determined based on the symptoms of the infection or suspected infection or the general clinical condition of the subject. Although any microbial infection is included, the methods of the present invention are of particular use during the detection or diagnosis of sepsis (or more generally in the treatment of sepsis) or when sepsis is suspected. Thus, a clinical or veterinary sample can be from a subject suffering or suspected of suffering from sepsis or at risk of suffering from sepsis. In this case, the sample is usually blood or a blood-derived sample. Typically, for sepsis, the sample will be or will contain blood, but other types of samples, such as those listed above, are not excluded.

The clinical sample can be introduced into the culture vessel containing the culture medium. This is a standard procedure that can be performed according to standard procedures well known in the art and widely described in the literature. A clinical sample can thus be cultured, and thus the sample used in the method can be a culture of a clinical sample (or, accordingly, a veterinary sample). The following discussion is in the context of clinical samples, but it should be understood that this may similarly refer to veterinary samples.

Clinical samples can be collected into containers containing media suitable for culturing microbial cells. In some embodiments, it may be desirable to introduce a clinical sample into a culture flask and remove an aliquot of the clinical sample/medium mixture from the flask for testing (eg, for microorganisms) immediately or only after a short period of incubation ID), and the flasks were continued to grow until further testing (eg, AST testing). Such a method is described in WO 2015/189390.

Culture vessels can include any vessel or vessel suitable for culturing microbial cells, such as plates, wells, tubes, bottles, flasks, and the like. Conveniently, where the clinical sample is blood or a blood-derived sample, the culture vessel is a blood culture bottle, or indeed any tube, flask or bottle known to be used for blood sampling, especially for culturing for the purpose of detecting microorganisms . Thus, the sample can be a blood culture sample.

Conveniently, the culture vessel may be provided with a medium already contained therein. However, the culture medium can be provided separately and introduced into the culture vessel before, at the same time as, or after the addition of the clinical sample.

The culture medium may be any suitable culture medium and may be selected according to the nature of the clinical sample and/or the suspected microorganism, and/or the clinical condition of the subject from which the sample is obtained, and the like. Many different microbial media suitable for this use are known. As is known in the art, the medium can generally contain sufficient nutrients to promote rapid growth of the microorganisms. In many cases, suitable media are complex growth media, including media such as the following: Muller-Hinton (MH) medium, MH-fastidious (MHF), Muller-Hinton medium supplemented with lysed horse blood , Lysogeny Broth (LB), 2X YT Medium, Tryptic Soy Broth (TSB), Columbia Broth, Brain Heart Infusion (BHI) Broth and Brucella Broth and as known in the art A general-purpose growth medium, and can include the addition of specific growth factors or supplements. Cultures can be agitated or not. The medium can be obtained in a variety of forms including liquid, solid and suspension, etc., and any of these can be used, but conveniently the medium is a liquid medium. Where the culture vessels are ready-to-use blood culture bottles as described above, these vessels may contain specific media that have been specially modified to allow the growth of a wide range of microorganisms. Typically, the medium provided by the manufacturer in the blood culture bottle will contain reagents or additives to neutralize the presence of any antibiotics present in the clinical sample taken from the test subject. Flasks with or without such neutralizing reagents can be used, and neutralizing reagents can be added to the culture vessel if desired.

In a particular aspect of the invention, the clinical sample is blood or a blood-derived sample and is collected in a blood culture bottle (BCF). Examples of blood culture bottles include BacT/ALERT (Biomerieux) blood culture bottles, Bactec blood culture bottles (Becton Dickinson) or VersaTrek blood culture bottles (Thermo Fisher), or indeed known for blood sampling, especially for culturing and Any tube, flask or bottle for the purpose of detecting microorganisms.

Such blood culture bottles and the like may contain resin, and the method may accordingly include a step of removing the resin from the sample, eg by filtration. For example, such a resin pre-filtration step may be performed prior to performing step (b) of the method.

The samples according to the invention may accordingly comprise clinical samples in the culture medium. Additionally, the sample may be a clinical sample culture (ie, a clinical sample that has been cultured for a period of time). In this regard it can be seen that the sample subjected to the method of the present invention may be part of a collected or prepared composite sample. Thus, in one embodiment, the sample of the method of the invention may be from a sample, for example from the contents of a culture vessel (flask) containing a clinical or other sample (whether before, during or after a period of incubation (ie incubation) ) taken or removed from aliquots (eg test aliquots).

Thus, in one embodiment, the sample provided in step (a) may be a culture of a clinical sample that has been designated as positive for microbial growth (eg, in a clinical sample culture system). Thus, it can be a positive blood culture bottle. However, according to the method of the present invention, clinical sample cultures need not be designated as positive, and can be used at a stage before the clinical culture sample is designated as positive, eg when it has been cultured for less than the period of time necessary to designate positive Such clinical culture samples. Thus, the sample may be a non-positive blood culture bottle (eg, a blood culture bottle that contains fewer microbial cells than is required to designate a flask as positive, or a blood culture bottle that has been cultured for a shorter period of time). Indeed, in the case of some clinical samples, a sample of the clinical sample culture can be removed and used in the methods of the invention prior to any culturing (eg, when establishing a clinical sample culture).

Certain microorganisms are known to be difficult to culture and, in clinical situations, may not be detectable in clinical detection or diagnostic methods based on the culture step by traditional or routine methods. For example, some bacteria are difficult to grow on solid media commonly used in diagnostic methods. Thus, the number of clinically relevant microorganisms may far exceed the number of microorganisms commonly tested and analyzed today. Such "unculturable" microorganisms (eg, bacteria) that may not be able to use standard culture methods can be grown, for example, in certain liquid media with various supplements or additives (eg, serum or other blood components or BHI, etc.). However, such supplements or additives may interfere with concentration determination methods and may need to be removed. The methods disclosed herein may be applicable in this context, and the sample may accordingly be a sample of a culture of such a microorganism. Microorganisms can be present in clinical or veterinary samples that have been cultured (eg, in specialized media containing supplements or additives). However, in another embodiment disclosed herein, the culture may be an isolate of a microorganism (eg, an isolate from another culture), and thus in this case the sample may not necessarily contain mammalian cells. Such samples can be used in the context of the methods described above for determining the concentration of microorganisms in suspension (ie, methods that do not include the steps of providing a sample comprising microorganisms and mammalian cells and recovering the microorganisms therefrom).

In a method comprising recovering microorganisms from a sample comprising microorganisms and mammalian cells, the sample is contacted with a buffer solution, a detergent, and one or more proteases. Contact of the sample with these reagents causes lysis of mammalian cells present in the sample. The reagent caused lysis of mammalian cells, but not microbial cells. In particular, the reagents do not cause lysis of bacterial cells. Preferably, the agent also does not cause lysis of fungal cells; preferably, the agent also does not cause lysis of non-mammalian eukaryotic microbial cells (eg, protists). The reagents generally work by solubilizing mammalian cell membranes. Selective lysis of non-microbial cells allows microbial cells to be separated from other components that may be present in the sample. The term "lysis" refers to cell rupture. Specifically, cells rupture to release cellular contents. The term "selective lysis" or "selective lysis" refers to the lysis of a specific subset of cells present in a sample. In the present case, it is desirable to selectively lyse only non-microbial cells present in a clinical or veterinary sample, or more particularly cells derived from a test subject (eg mammalian cells), without substantially lysing the non-microbial cells present in a clinical or veterinary sample Microbial cells in clinical or veterinary samples. In addition, according to certain methods of the present invention, it is desirable that the microbial cells obtained from the sample be able to grow and reproduce (required for growth in order to determine antimicrobial susceptibility), therefore it is desirable that the microbial cells have the ability to grow and/or reproduce (viability) not Influenced by selective lysis of non-microbial cells present in the sample or cells derived from the test subject.

Preferably, after the selective lysis of mammalian cells, all (ie 100%) or substantially all of the microbial cells present in the sample remain intact, or more particularly, viable, and preferably after the selective lysis step , at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91% or 90% of the microbial cells in the sample remain intact or alive. However, since the method of the present invention requires determination of the concentration of intact or viable microorganisms in the recovered microbial sample, at least 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of the microorganisms Antibiotic susceptibility can still be assessed while cells remain viable. Thus, such methods are not limited to any particular level of microbial viability following selective lysis of mammalian cells.

The pH of the buffer solution is at least pH 6 and at most pH 9, ie the pH of the buffer solution is in the range of pH 6 to pH 9. In particular embodiments, the pH of the buffer solution is in the range of pH 6.0 to pH 8.5, pH 6 to pH 8, pH 6.5 to pH 8.0, or pH 7 to pH 8. Optimally, the pH of the buffer solution is about 7.5.

The buffer solution may contain chaotropic or chaotropic agents to increase lysis of target cells (ie, mammalian cells), such as urea, guanidine hydrochloride, lithium perchlorate, lithium acetate, phenol, or thiourea. However, in certain embodiments, the buffer solution does not contain a chaotropic agent or a chaotropic agent. In certain embodiments, such reagents may not be used during the recovery of microorganisms from the sample (and more particularly during the selective lysis step) and/or during the concentration determination of the methods of the invention .

The buffer solution preferably does not contain alcohol. The buffer solution may further contain reducing agents (eg, 2-mercaptoethanol or disthreitol (DTT)), stabilizers (eg, magnesium or pyruvic acid), humectants, and/or chelating agents (eg, ethylenediaminetetraacetic acid (EDTA) ).

Additionally, the buffer solution may contain any suitable salt, including _NaCl , _KCl , _MgCl2 , _KH2PO4 , _K2HPO4 , _{Na2HPO4 ,} and _{NaH2PO4 .} Such salts may facilitate mammalian cell lysis or subsequent processing of microbial cells. If present, the salt may be present in any suitable concentration, eg at least 0.01M, 0.02M, 0.05M, 0.1M, 0.2M, 0.5M, 1M, 2M or 5M, depending on eg the buffer used and the volume of the sample And other factors.

In a specific embodiment, the buffer solution is PBS buffer (phosphate buffered saline). PBS contains disodium hydrogen phosphate (Na ₂ HPO ₄ ), NaCl and optionally KCl and/or potassium dihydrogen phosphate (KH ₂ PO ₄ ). PBS can be obtained from manufacturers such as Sigma-Aldrich or Thermo Fisher Scientific, or can be readily prepared from its components. An exemplary formulation for 1x PBS is NaCl 137 mM, KCl 2.7 mM, _{Na2HPO4 10} mM, _KH2HPO4 1.8 mM _; pH can be adjusted up or down with NaOH or HCl, respectively.

The concentration of the buffer solution added to the sample can be higher than its use concentration, for example, the buffer solution can be added at a 5x or 10x concentration to dilute it to its use concentration when mixed with the sample.

The detergent may be an ionic detergent, a non-ionic detergent or a zwitterionic detergent. Ionic detergents are charged, which can be positively charged (cationic detergents) or negatively charged (anionic detergents). Zwitterionic detergents have multiple charged groups; typically, zwitterionic detergents have the same number of positive and negative charges, and thus have a net zero charge. Nonionic detergents have uncharged hydrophilic head groups.

Exemplary ionic detergents that can be used include alkyl benzene sulfonates, N-lauroyl sarcosine, deoxycholic acid (or a salt thereof, such as sodium deoxycholate), cetrimonium bromide (CTAB), and dodecane Sodium sulfate (SDS).

Exemplary zwitterionic detergents that can be used include CHAPS, sulfobetaines (eg, SB 3-10 and SB 3-12), aminosulfobetaines (eg, ASB-14 and ASB-16), and C7BzO.

Preferably, the detergent is a non-ionic detergent. Exemplary non-ionic detergents that can be used include the Triton detergent series (eg Triton X100-R and Triton X-114), NP-40, Genapol C-100, Genapol X-100, Igepal CA630, Arlasolve 200, Brij detergents series (eg Brij-O10, Brij-97, Brij-98, Brij-58 and Brij-35), octyl β-D-glucopyranoside, polysorbates (eg polysorbate 20 and polysorbate 80) , and the range of Pluronic detergents such as Pluronic L64 and Pluronic P84. In one embodiment, polyoxyethylene detergents can be used. The polyoxyethylene detergent may comprise the structure _C12-18 / _E9-10 , wherein C12-18 represents a carbon chain length of 12 to 18 carbon atoms and E9-10 represents 9 to 10 oxyethylene hydrophilic head groups . In a specific embodiment, the detergent is Brij-O10, which can be obtained, for example, from Sigma-Aldrich (product P6136). Brij-O10 has the following chemical formula:

wherein n is about 10, preferably 10.

Add detergent to the appropriate resulting concentration. Such concentrations are known to the skilled person or can be determined by routine optimization for any selected detergent. In a particular embodiment, the detergent is contacted with the sample at a concentration between 0.1% and 5% w/v, eg, between 0.1% and 1% w/v (ie the resulting concentration after adding the detergent to the sample) . In a specific embodiment, the detergent is contacted with the sample at a concentration of about 0.45% w/v.

The protease can be any suitable protease. It can be an endopeptidase or exopeptidase, and it can use any proteolytic mechanism, for example it can be a serine protease, cysteine protease, aspartyl protease, metalloprotease, and the like. Exemplary proteases useful in the methods of the present invention include Type XXIII proteases, proteinase K, pepsin, trypsin, chymotrypsin, papain, elastase, and cathepsin. Preferably, the protease is an endopeptidase. In a specific embodiment, the protease is proteinase K. The skilled artisan can determine the appropriate concentration of protease for use in the method of the present invention depending on the sample, the protease used, and the like. For example, proteinase K can be used at final concentrations ranging from 20 to 200 μg/ml (eg, 50 to 150 μg/ml or 50 to 100 μg/ml). Preferably, proteinase K is used at a final concentration of about 50 to 80 μg/ml.

The sample may also be contacted with additional enzymes to aid in mammalian cell lysis in step (b), such as nucleases (eg, DNase or RNase), lipases, glycoside hydrolases (eg, neuraminidase), amylases, and the like.

In step (b), the sample may be contacted with a buffer solution, a detergent and at least one protease, respectively. Alternatively, the three components (buffer, detergent, protease) can be prepared in one or more combinations (eg, pre-prepared as a combined composition, or prepared at the time of use) prior to contact with the sample. The term "contacting" is used broadly herein to include any manner of contacting a sample with a reagent in any order. Thus, a sample can be added to a component (eg, a component already present in the reaction vessel) or a component can be added to a sample (eg, a sample already present in the reaction vessel). Three or any two of the three components can be pre-prepared as a combined composition to be contacted with the sample, or the components can be added sequentially (eg, to a reaction vessel) prior to contact with the sample. In a preferred embodiment, the detergent is provided in a lysis buffer comprising the detergent dissolved in the aforementioned buffer. At least one protease can then be added to the lysis buffer and the resulting composition added to the sample (and vice versa) such that the sample is contacted with the buffer solution, detergent and protease simultaneously. In a specific embodiment, the lysis buffer comprises PBS pH 7.5, 0.45% w/v Brij-O10. In a specific embodiment, the sample is contacted with a composition comprising: (i) a lysis buffer comprising PBS pH 7.5 and 0.45% w/v Brij-O10 and (ii) proteinase K.

The contacting of step (b) (ie, contacting (or incubating) the sample with buffer solution, detergent, and one or more proteases) is performed for an appropriate time. For example, the contacting can be carried out for up to 1 hour, such as up to 30 minutes, up to 20 minutes, or up to 10 minutes. The contacting is carried out at a suitable temperature, at least 4°C, eg 20-40°C, eg 25-37°C. The aliquot can be heated for 5-20 minutes, preferably 5-10 minutes.

The mixture obtained in step (b) is filtered. The filtration process allows for the separation of intact microbial cells from the mammalian cell lysate and optionally any other debris or material present in the sample. Intact microbial cells are captured in the filter, while mammalian cell lysate is passed to discard, thereby removing lysed mammalian cells from suspension. Filtration is carried out using a filter including suitable pore size to capture any microbial cells. The pore size of the filter may be 0.5 μm or less; preferably, the pore size of the filter is 0.25 μm or less. The filter may be made of any suitable material, for example many suitable filters are made of PTFE (polytetrafluoroethylene). Suitable filters are commercially available, for example, from Merck. In some embodiments, the filter used has a larger surface area relative to the volume of the sample filtered through it to prevent clogging of the filter by microorganisms. For example, the size of the filter may range from 30-100, 30-80mm or 30-75mm (eg 50mm). However, any size filter can be used, for example in the 4-100, 4-80 or 4-75mm range. This may depend on the nature of the sample and the amount of microorganisms in the sample. For example, positive blood cultures may contain more microorganisms than clinical urine samples, and larger filter sizes may advantageously be used. Appropriate filter sizes can be determined by routine trial and error.

After filtration, the isolated microbial cells (ie, those trapped on or inside the filter) can be washed to remove residual lysis buffer, mammalian cell debris, and the like. If so, washing is performed between steps (c) and (d). Washing can be performed by flushing wash buffer through the filter. The filter can be washed with any suitable wash buffer, as known to the skilled person. Suitable wash buffers include, for example, buffer solutions as described above, eg, PBS. In a specific embodiment, the wash buffer can be a buffer solution as described above, and in certain embodiments can be the same buffer solution used in step (b). However, in other embodiments, the wash buffer may contain protease (and optionally no detergent) or detergent (and optionally no protease). In certain embodiments, the wash buffer may contain a chaotropic agent, while in other embodiments, it may not contain a chaotropic agent, eg, as described above. In additional embodiments, the wash buffer may be a culture medium as described above. In a specific embodiment, the wash buffer is cation-adjusted Mueller Hinton Broth (CAMHB), which is commercially available, eg, from Sigma-Aldrich. CAMHB may also be called Mueller Hinton Broth 2. The filter (containing the isolated microbial cells) can be washed one or more times as needed to remove mammalian cell debris from the filter, eg, the filter can be washed 2, 3, 4 or 5 times or more.

After filtration and optional washing, the microbial cells are recovered from the filter. Recovery of microbial cells involves resuspending the cells in a liquid, thereby providing a suspension of recovered microorganisms. Cells from the filter surface can be resuspended using liquid by repeated pipetting. In a preferred embodiment of the present invention, the liquid is backflushed through the filter (ie, in the opposite direction to filtering the filtrate) to resuspend the microbial cells. In another embodiment, the microorganisms are recovered by drawing back the last portion of the wash solution through the filter. Alternatively, the microbial cells can be recovered by using the entire filter, for example by adding liquid to the filter or contacting the filter with the liquid in the container.

The liquid in which the microbial cells are resuspended can be any suitable liquid, such as a buffer or culture medium. In a preferred embodiment, the microbial cells are resuspended in a medium (ie, a liquid growth medium suitable for culturing the microorganism). When using a medium to resuspend microbial cells, in general, the medium is one that is approved or approved for use in AST assays. In one embodiment, it is Muller-Hinton (MH) medium or Muller-Hinton Fastidious (MHF) medium, or cation-regulated Mueller Hinton medium (CAMHB). For non-standard AST, any other commonly known medium can be used in the present invention. MIC values obtained by performing AST assays using "non-standard" media can be adjusted (correlated) to yield standard AST results. In other embodiments, the resuspension liquid may be PBS or other buffers. In other embodiments, the resuspension liquid is not water (eg, tap water, ground water, or sterile water). Furthermore, in certain embodiments, the liquid in which the microbial cells are resuspended may not contain proteolytic enzymes such as papain, trypsin, neutral protease, subtilisin or subtilisin-like enzymes or Rhozyme.

Once the microbial cells have been recovered and the recovered microbial sample has been obtained, the concentration of microbial cells present in the recovered microbial sample is determined according to the method of the present invention. As mentioned above, in a particular embodiment, this is especially for the purpose of performing an AST assay, ie, the concentration of microorganisms can be determined prior to performing an AST assay.

Advantageously, using recovered microbial samples for AST assays may allow for faster AST assays. In particular, by recovering microbial cells directly from a clinical sample or clinical sample culture, thereby obtaining a recovered microbial sample, a homogeneous sample free of any contaminants is provided.

Certain samples, such as food or environmental samples in particular, may contain particulate matter, which may wish to be removed prior to determining the concentration of intact microorganisms in the sample. Additionally, certain commercially available culture vessels (eg, blood culture bottles) are provided with resin beads that neutralize the action of any antimicrobial agents present in clinical samples (ie, those that have been administered to test subjects), to promote the growth of microbial cells in culture. Thus, in a preferred embodiment, the sample can be filtered to remove any large particles that may be present in the sample. Preferably, this filtration step will be performed using a filter having a pore size (eg 100, 200 or 300 μm, but can be as high as 1000 μm) that does not substantially remove any cellular material from the test aliquot, but can remove particles. Such filtering steps can be performed at any point in the method of the present invention. In certain embodiments, this step may be performed prior to imaging the suspension-stain mixture in step (e)(iv) to avoid any such particles from being imaged. Thus, such a step may be performed before step (e)(iii) or step (e)(i), and more particularly may be performed before step (e). More particularly, such a step may be carried out before step (c) or step (b), and still more particularly, may be carried out before step (a). In certain embodiments, the sample provided in step (a) may have undergone such a filtering step to remove particulate matter. To determine the concentration of intact microbial cells in a suspension, the suspension is first aliquoted, ie divided into one or more smaller fractions/samples. An aliquot (ie a portion) of the suspension is first processed (in step (e)(i)) to enhance the staining process. The treating (or "pre-treatment") step can include contacting an aliquot with an alcohol (eg, ethanol). Other suitable alcohols include methanol, propanol, isopropanol, butanol (in any isomeric form), and the like. Those skilled in the art will be able to select a suitable alcohol. In a preferred embodiment, an aliquot is contacted with ethanol. In certain embodiments, an aliquot is contacted with an alcohol to provide an alcohol comprising 25-45% v/v alcohol, eg, 25-35% v/v alcohol, 30-40% v/v alcohol, or 30-35% alcohol Mixtures of v/v alcohols such as ethanol. In a specific embodiment, an aliquot is contacted with an alcohol to provide a mixture comprising 30% v/v alcohol (eg, ethanol). In another specific embodiment, an aliquot is contacted with an alcohol to provide a mixture comprising 35% v/v alcohol (eg, ethanol).

In an alternative embodiment, the processing step includes heating an aliquot of the suspension. The aliquot can be heated to a temperature in the range of 50-90°C, eg, 60-80°C or 65-75°C. In a specific embodiment, the aliquot is heated to a temperature of about 70°C. The aliquot can be heated for an amount of time appropriate to the temperature used, ie, the higher the temperature selected, the shorter the heating time required (and vice versa). In one embodiment, the aliquot is heated for 30 seconds up to 20 minutes, or up to 10 minutes. The aliquots can thus be heated for 0.5-20 or 0.5-15 or 0.5-10 minutes (the time measured is the time at the relevant temperature, ie not the ramp-up time). The skilled person can select an appropriate heating time for a given heating temperature. Heating can be performed, for example, in an incubator, heat block, oven, thermal cycler, or any other suitable device.

In certain embodiments, the alcohol treatment can be combined with the thermal treatment steps simultaneously or separately (eg, sequentially).

In another alternative embodiment, the treating step comprises contacting an aliquot of the suspension with a detergent. Suitable detergents are described above with respect to the lysis buffer for step (b). When contacting the suspension of microbial cells with the detergent in step (e)(i), the detergent described in step (b) can be used, but at a much higher concentration than that used in step (b). Thus, while the detergent in the buffer solution of step (b) may be present as described above at a concentration of, for example, between 0.1% and 5% w/v, for example, between 0.1% and 1% w/v, step (e) ( The detergent used in i) is used at a much higher concentration, preferably 5-20 times, eg 10 times. The detergent may be used in step (e)(i) at a concentration of 0.5% to 50% w/v, preferably 1% to 10% w/v, eg about 5% w/v.

In embodiments where an aliquot of the suspension is treated with an alcohol or detergent, the treatment may be performed at or near room temperature, eg the treatment may be in the range of 20-37°C, eg, 20-30°C, 25-30°C Or 30-35 ℃ temperature. Alternatively, as described above, this can be combined with a heating step. Contacting can be carried out by incubating with a selected concentration of alcohol or detergent at a selected temperature. Incubation may last from 30 seconds to as long as 1 hour, eg, as long as 30 minutes, as long as 20 minutes, as long as 10 minutes, or as long as 5 minutes. The precise time will depend on the sample, the microorganisms present in the sample and/or whether a heat treatment step is included. In a preferred embodiment, the incubation lasts 5 to 10 minutes, preferably about 5 minutes.

In certain embodiments, the processing step does not include contacting the sample with an aldehyde or ketone. In particular, the processing step may not include contacting the sample with formaldehyde, ethanol, propionaldehyde, acetone, butyraldehyde, or butanone. In additional embodiments, the treating step does not include contacting the sample with a carboxylic acid, such as formic acid, acetic acid, oxalic acid, propionic acid, malonic acid, butyric acid, or succinic acid. In additional specific embodiments, the processing step does not include contacting the sample with an aldehyde, ketone or carboxylic acid (eg, as listed above) in combination with a thermal treatment step, and/or in combination with contacting the sample with an alcohol and/or detergent. In further embodiments, the treatment step does not include contacting the sample with antibiotics, particularly antibiotics that can allow bacterial growth but inhibit cell division, such as chloramphenicol and penicillins, such as ampicillin, benzyme penicillin, chloramphenicol Cloxacillin, dicloxacillin, or a combination thereof.

The samples analyzed by the method of the present invention can contain a wide range of possible concentrations of microorganisms, and it is not possible to make a single calibration curve to allow accurate determination of such concentration ranges. Therefore, it may be beneficial to dilute an aliquot of the sample containing microorganisms during the performance of the method of the invention such that the image analysis value for the number of objects determined in step (e)(iv) falls within the range of the predetermined calibration curve. scope.

Furthermore, depending on the nature of the suspension and/or processing, it may be desirable to dilute the sample (ie an aliquot of the suspension taken to allow concentration determination in step (e)) to allow for concentration determination, for example it would be possible to The dilution (or minimization or reduction of its amount) of contaminants or components that interfere with the method of concentration determination. For example, certain media (eg, Muller Hinton's medium) contain components that may interfere with fluorescence assays, and if the sample is a sample of a culture containing such a medium, or if the recovered microorganisms are resuspended in such a medium , a dilution step may be desired. Likewise, if alcohol or detergents are used for treatment, a dilution step may be desired. Alternatively, if the microorganisms are resuspended from the filter in a buffer (eg PBS), a dilution step or, more particularly, an initial dilution step may not be necessary. It may be relevant in the case of a method in which the microorganisms are present in suspension in low concentrations (low amounts), in which case it may be desirable to resuspend the recovered microorganisms in a buffer (eg PBS).

When dilution is performed, ie when an aliquot of the sample is diluted in step (e)(ii) to provide a diluted aliquot at the dilution value, such dilution may be before, during or after step (i) conduct. Thus, an aliquot of the sample may be diluted before, during or after the treatment in step (e)(i) prior to contact with the stain. In this case, the dilution medium may be a buffer, or saline or water or other aqueous solution, etc., as will be discussed in further detail below.

In one embodiment, no dilution is performed prior to step (e)(i) (ie, prior to contact with the alcohol (or detergent) or heating). In other words, the dilution can be performed during or after step (e)(i).

In another embodiment, where the "pretreatment" of step (e)(i) involves contacting with an alcohol or detergent, the contacting itself may provide a dilution step. This can be seen as a dilution step during step (e)(i).

In other embodiments, the methods herein may include a dilution step after the contacting/heating of step (e)(i), eg, after contacting with an alcohol.

In a particular embodiment, the method may comprise performing the dilution of step (e)(ii) during and after step (e)(i). For example, dilution of an aliquot may be performed during step (e)(i) during contact with alcohol, and further dilution may be performed after contact with alcohol.

Two or more aliquots can be prepared such that each aliquot is diluted to a different degree. In other words, each aliquot can be diluted with a different dilution factor or dilution value. In such embodiments, the first aliquot (ie, at the first dilution value) may be an aliquot of the sample, and the second aliquot (or subsequent aliquots) may be at the second (or subsequent) diluted aliquot of the dilution value. Alternatively, two separate dilutions can be performed. One or more diluted aliquots can be diluted by serial dilution. Thus, dilution series can be prepared by a series of sequential, separate or simultaneous steps as desired.

When heat treatment of aliquots is used in step (e)(i), if dilution of the suspension is required, the dilution may be performed before, during, or after heating (ie, the treatment at (e)(i) may be performed The dilution of step (e)(ii) is performed before, during or after step. However, if an alcohol or detergent is used to treat the aliquot, in one embodiment, dilution of the aliquot is preferably performed after the treatment step of (e)(i) to dilute the alcohol or detergent and thereby Enhanced staining/imaging process. In particular, ethanol may interfere with the staining process of the claimed method, so preferably, if ethanol is used to treat an aliquot of the suspension, it is diluted to reduce the ethanol concentration prior to imaging.

In certain embodiments of the invention, when two or more aliquots are prepared, it may be simultaneously (or substantially simultaneously) prior to step (e)(iii) of contacting with the colorant, including by sequential or successive steps) to prepare each of the aliquots. In this case, steps (e)(iv) and (e)(v) can be performed simultaneously (ie, in parallel) or sequentially for each aliquot. In other words, each aliquot can be imaged simultaneously (ie, in parallel) or sequentially, and the individual image analysis values obtained from each aliquot can be compared to a predetermined calibration curve. Alternatively, steps (e)(iv) and (e)(v) may be performed on a first aliquot, and if the image analysis values obtained from said aliquot fall within the range of a predetermined standard calibration curve , then steps (e)(iv) and (e)(v) may be omitted for the second or additional aliquot. The aliquot may be an aliquot of the treated suspension of step (e)(i), or a diluted aliquot of step (e)(ii).

However, in alternative embodiments, only the first aliquot (which may be an aliquot of the treated suspension of step (e)(i), or a dilution of step (e)(ii) A diluted aliquot (or a second or additional diluted aliquot) is not prepared until the steps of the method are performed. Such an embodiment may be desirable if, for example, the image analysis values do not fall within the range of the predetermined calibration curve. In such embodiments, it may be necessary to repeat the method of the invention on a second (or additional) aliquot at a different dilution value. In this case, it will be seen that each of the two (or additional) aliquots is prepared in sequence, i.e. after step (e)(iv) has been performed on eg the first aliquot and/or after (e)(v).

Thus, (e)(iv) and/or (e)(v) may be for one aliquot (which may be pretreated, but diluted or undiluted) even if more than one aliquot is prepared aliquots), or on two or more aliquots (which may be diluted aliquots, or may include undiluted aliquots).

Thus, steps (e)(iii) and (e)(iv) can be performed on each of two or more aliquots to determine the number of viable microorganisms in each aliquot corresponding to the The image analysis value of the number of objects. When the two or more image analysis values for each of the two or more aliquots have been obtained, step (e)(v) may include identifying images comprising images within a predetermined calibration curve range An aliquot of the values is analyzed, and the image analysis value of the aliquot is subjected to a predetermined calibration curve to determine the concentration of microorganisms in the sample. In this case, steps (e)(iii) and (e)(iv) may be performed sequentially or simultaneously on each aliquot. As mentioned above, the aliquot can be a diluted aliquot, or it can include an undiluted aliquot.

Dilution can include contacting an aliquot of the sample with a volume of a suitable sterile buffer or aqueous solution (eg, saline or saline) or virtually any suitable diluent. The aliquot can be diluted using the same liquid (eg, culture medium) used to form the microbial suspension in step (d). Preferably, a buffer is used to dilute an aliquot of the suspension. The buffer may be any buffer known in the art such as PBS, HBS (HEPES buffered saline), Tris buffer (eg Tris-HCl) or TBS (Tris buffered saline) or MOPS buffer. In a preferred embodiment, an aliquot of the suspension is diluted with PBS.

If the aliquot is treated with heat or alcohol in step (e)(i), the diluent may comprise a detergent. In terms of the type and concentration of detergent, the detergent may be as described above for the lysis buffer of step (b). Using a low concentration of detergent in the diluent helps to calculate the concentration of microorganisms by separating bacterial clusters, which facilitates image analysis.

An aliquot of the treated and optionally diluted suspension is then contacted with the stain to provide a suspension-stain mixture. The stains used in the methods of the present invention are fluorescent stains capable of binding DNA. The stain can be cell permeable or cell impermeable. "Cell-permeable" refers to an agent that is capable of passing through the intact membrane of living cells. "Cell-impermeable" refers to an agent that cannot pass through the intact membrane of living cells. Without being bound by theory, it is believed that the treatment of the cells in step (e)(i) disrupts their cell membranes (and where relevant cell walls) without lysing the cells. Thus, after treatment, cell-impermeable stains are able to enter and stain cells, as are cell-permeable stains, of course. Fluorescent stains have an emission wavelength that can be detected using a fluorescence detector, thereby enabling the identification of stained cells.

Certain stains capable of binding to DNA are also known to exhibit enhanced fluorescence when bound to DNA compared to when present free in solution. Preferably, the selected fluorescent dye exhibits this property. In other words, in a preferred embodiment, when the dye binds to DNA, the fluorescence intensity of the dye increases. Selecting a stain with this property may help reduce the level of background signal generated at the emission wavelength during detection. In particular, dyes can be selected that have low fluorescence when not bound to DNA (ie, when free in solution). For example, the stain may exhibit less than 50%, more preferably less than 40%, 30%, 20% or 10% fluorescence, or more preferably, when free in solution, compared to the fluorescence displayed when bound to DNA less than 10% fluorescence, such as less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% fluorescence, or less than 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% fluorescence.

The dyes may have excitation and emission wavelengths of 350-700 nm wavelengths. A range of suitable fluorescent dyes having emission wavelengths within this range are known in the art, and exemplary fluorescent dyes are described below. The fluorescent dye may be a green fluorescent dye, ie, having a peak fluorescence emission intensity at or near light having a wavelength of 510 nm. In a preferred embodiment, the stain is a cell permeable stain.

Particularly preferred stains with all of the above desirable properties include SYTO green fluorescent nucleic acid stain (Molecular Probes). SYTO dyes are examples of asymmetric cyanine dyes, and thus, asymmetric cyanine dyes can be preferably used as dyes in the method of the present invention. Structures of useful SYTO dyes are provided in US US5658751, US6291203, US5863753, US5534416 and US5658751. Many different SYTO stains are available, including SYTO 9, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 16, SYTO 21 and SYTO 24, which can be used in the methods of the present invention. Particularly preferred are SYTO 9 and/or SYTO 13 or SYTO BC (mixtures of dyes). The SYTO BC stain mixture has excitation wavelengths of 473-491 nm and emission wavelengths of 502-561 nm.

Alternatively, the fluorescent stain may be a cell-impermeable stain, which may be red fluorescent, ie, have a peak fluorescence emission intensity at or near light having a wavelength of 650 nm. A preferred red fluorescent dye suitable for use in the method of the present invention is propidium iodide (PI).

However, the stain can be any fluorescent stain capable of staining nucleic acids. These can include SYBRGreen, SYBR Gold, SYBR Green II, PicoGreen, RiboGreen, DAPI, Hoechst 3342, Vybrant dyes, etc., or almost any dye commercially available from Thermo Fisher. See, eg, the Thermo Fisher website ( https://www.thermofisher.com/se/en/home/references/molecular-probes-the-handbook/nucleic-acid-detection-and-genomics-technology/nucleic-acid-stains . Dyes mentioned in Section 8.1 (Nucleic Acid Stains) of the Molecular Probes Handbook in the Technical Reference Library available at html ), which is incorporated herein by reference.

The aliquots can be contacted with the stain at a temperature that is not detrimental to the cells in the suspension-stain mixture and allows staining to occur. Suitable temperatures can be selected, for example, based on the nature of the sample, the identity of the microorganisms therein, or the nature of the stain used. However, to avoid damaging the microorganisms in the sample, temperatures of 37°C or lower are usually used. Thus, temperatures of 35°C, 30°C or 25°C or lower can be used. It is also preferred to use temperatures of 4°C or higher, such as 5°C, 10°C or 15°C or higher. In a preferred embodiment, the sample is contacted with the stain at 20-30°C, more particularly at 20-25°C. In certain embodiments, the sample can thus be contacted with the stain at room temperature.

Objects are identified as corresponding to intact microorganisms by detecting the fluorescent signal at the emission wavelength of the dye. Thus, objects corresponding to intact microbial cells have different fluorescence properties than other objects in the sample and can be distinguished from other objects in the sample (eg, objects corresponding to non-intact microorganisms, cell debris, or other particles present in the sample) open, allowing the number of objects corresponding to intact microbial cells to be determined. Preferably, only stained microorganisms corresponding to intact microorganisms in the sample are fluorescent and no other objects are detected during fluorescence imaging.

Imaging of the suspension-stain mixture is performed by visual inspection means. Magnified images of the suspension-stain mixture were obtained and analyzed to detect objects corresponding to intact microorganisms.

Although an object corresponding to an intact microorganism may be a microbial cell, which may or may not be intact after pretreatment, it may also be a cluster of two or more cells, such as clones grown into clusters and/or Aggregates of non-clonal cells. Thus, the object can be a microbial cell or a cell cluster. Different microorganisms may grow in different ways, eg aggregated or non-aggregated, or have different patterns or morphologies, and for a given microorganism this may also vary according to growth conditions (eg presence or amount of antimicrobial agent). Such different growth patterns and/or morphologies, etc. can be considered by analyzing the images and counting the objects and then correlating the number of objects with the concentration of microorganisms. Thus, images can be analyzed by counting the number of objects and adjusting the number based on, for example, the size and/or intensity of the objects (eg, to account for cell clusters or cell aggregates) to provide an image analysis value for the number of objects, which can then be used using A calibration curve correlates this to the concentration of intact microorganisms. As described above, low concentrations of detergent can be added to sample aliquots to reduce aggregation.

Imaging of suspension-chromosome mixtures can be performed at temperatures that are not harmful to microorganisms. Typically, this will be carried out at room temperature or 20-25°C, although other temperatures can also be used, such as temperatures of at least 4°C to 37°C (ie, 37°C or lower).

Imaging is performed at the emission wavelength of the dye, i.e. the object stained with the fluorescent dye is detected. As mentioned above, this provides sufficient information to allow objects corresponding to intact microorganisms to be distinguished from other objects that may be present in the sample.

In addition to fluorescence, imaging can include the use of microscopy, including brightfield, tilted field, darkfield, dispersion staining, phase contrast, differential interference contrast, confocal microscopy, single plane illumination, light sheeting, and/or widefield Multiphoton Microscopy.

Microorganisms can be allowed to contact, bind, associate or adsorb onto a detection surface for imaging. However, in a preferred embodiment, imaging is performed on a suspension of microorganisms, ie microorganisms in a suitable medium or buffer, rather than microorganisms attached to or immobilized on or at a surface. In other words, a volume of suspension-stain mixture can be imaged. In the case of imaging a suspension of microorganisms, the images may be obtained at one or more focal planes through the suspension. It may be preferable to obtain images at two or more (different) focal planes through the suspension (eg, at different depths or cross-sections through the suspension-stain mixture). In other words, separate sub-volumes of the volume to be imaged can be imaged (ie, images of separate sub-volumes of the volume of the suspension-stain mixture can be obtained). Alternatively, the images may be acquired at different locations, eg at different locations in the sample chamber, eg at different X-Y locations in the sample chamber with low height. In such an arrangement, the majority of microorganisms will be in a single focal plane at each location. Thus, multiple (ie, two or more) non-overlapping images can be obtained. Such a plurality of images may include at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 or more images. The images are analyzed to detect and/or identify objects corresponding to microorganisms, which can be used to represent or indicate the presence of intact microorganisms in suspension, as described above. Thereby, image analysis values for the number of objects are obtained. Objects detected in all images of the suspension obtained can provide the total number of objects in the suspension.

To perform the imaging step, the suspension-stain mixture of step (e)(iii), or a portion or aliquot thereof, is provided (eg transferred to) in a container or vessel, eg suitable for imaging, in which imaging may be performed the wells of the plate or the compartments of the carrier. Such wells or compartments will have an optical viewing area or space, ie a viewing (or observable) area or space that is accessible to the microscope (or, more specifically, its objective), and of optical quality that allows microscopy and imaging. The geometry of the aperture/compartment may give an observable area of defined or desired size (eg, at least 2mm by 2mm) with a suitable or desired liquid height to allow imaging of the volume (eg, at least 2mm of liquid height). The objective lens can be focused on a plane inside the aperture or compartment, e.g. parallel to and at a distance from the bottom (e.g., about 0.1-0.5, e.g., 0.2 mm from the bottom), and the microscope can be configured to focus during imaging The plane is moved continuously through the liquid (eg, upwards by the suspension), eg, a total of 1-3 mm (eg, 1.5 mm) during image acquisition (eg, 10-60 or 20-30 seconds).

In a particularly preferred embodiment, imaging may comprise acquiring a series of 2-D images along the optical axis, wherein each image is acquired at a different location along the optical axis through the suspension volume. In certain embodiments, each image may be aligned perpendicular to the optical axis (referred to herein as xy alignment). Specific areas of the aliquot-sample mixture are covered in a single xy-aligned image, the size of which depends on the optical properties of the imaging device. For each location in xy space, one or more 2D images can be collected at different intervals along the optical or z axis. Thus, a series or stack of 2D images can be generated, which in one embodiment can be used to provide 3D information of the sample volume. Alternatively, multiple separate images that provide 2D information can be used. An alternative approach to extracting 3D information from a sample is that employed by Unisensor (see eg US8780181), where the optical axis is tilted relative to the xy plane and the sample or detector is moved along the x or y plane. Here, in addition to the xy space, a series of images extending into the z space are also acquired. Alignment of 2D image stacks perpendicular to the xy plane can also be achieved using this method with subsequent transformations of the image data. In this way, each image in the series of images is an image of a separate area (separate cross-section), or can alternatively be considered a separate volume (the cross-section has a defined volume in the z-direction, so can be used for each image provides a volume containing xy space of depth z).

Once extracted, the 3D information inherent in the 2D image stack can be used to identify objects in the sample that correspond to intact microorganisms. In one embodiment, a 2-D image can be generated from 3-D information by, for example, projecting the z-stack into a 2D image (projected 2D image). The resulting 2-D images can then be used for analysis. Alternatively, each image obtained by the volume of the suspension can be analyzed, and the results of the analysis can be integrated across all 2D images obtained on the sample. As another alternative, each of the various 2-D images obtained can be analyzed separately (ie, the objects in each 2-D image can be determined separately), and the information gathered therefrom can be combined . Objects can be determined as points or areas of fluorescence intensity that indicate intact microorganisms in the field of view under study, eg in an image or projected 2D image. Fluorescence images can be analyzed, and many alternative algorithms for this exist in, for example, Cellprofiler and most commercial image analysis systems.

In another embodiment, a change in intensity in z-space extending over each location in xy-space is recorded, which is indicative of microbial mass in a particular location. Integrated over the entire xy space, this gives a measure of total microbial volume. Algorithms for this program are also present in commonly available image analysis software, such as the freeware Cellprofiler.

Alternatively, the microscope can be configured to take images (eg, move the objective to), eg, in the X-direction (as opposed to the Z-direction), at different positions, eg, in the suspension-stain mixture (or field of view). the objective lens moves to).

Once an object corresponding to an intact microorganism (ie, an object detected at the emission wavelength of the fluorescent stain) has been detected by imaging, the information thus obtained can be used to generate image analysis values for the aliquot. The image can be analyzed for fluorescence intensity and/or size of the object (eg, each object), and optionally the morphology of the object (eg, each object). Such as the circularity of the object, the uniformity or maximum fluorescence intensity of the object's fluorescence intensity (eg the maximum intensity of a pixel in it), the modal fluorescence intensity in the object, the median or average fluorescence intensity and/or detected by imaging can be determined. factors such as the area of each object. In certain embodiments, only those objects having one or more of these parameters within a given range may be included in the analysis (eg, counted or enumerated), thereby generating image analysis values. The image analysis value may be a combined value of objects identified in the sense of representing or corresponding to the number of objects (ie, counts). Object area can be determined based on the number of consecutive pixels contained in each object, and only those objects containing at least or more than a certain number of pixels can be included in the analysis. In some embodiments, objects can be identified and detected based on a derived value for object area x intensity, and only those objects with properties that fall within a particular range of parameters can be counted or enumerated to generate image analysis value. In other words, the image analysis value represents the number of objects corresponding to intact microorganisms having features that fall within certain parameter ranges, or in other words, the corrected (or adjusted) number of objects corresponding to intact microorganisms.

Factors determined for each object (eg, any of the factors described above) or derived values such as object area x intensity for all objects may also be combined to provide information about the population of imaged objects (ie, about the total number of objects). In this way, for example, the maximum, mode or median fluorescence intensity of an imaged object (or more specifically, a collection or group of imaged objects) can be determined. Alternatively, the distribution of the fluorescence intensity of the imaged object, or a derived value such as object area x intensity, of the imaged object may be determined. Thus, each object may have a value assigned to it (eg, area, maximum fluorescence intensity, total intensity, median, or average intensity), and a median or median of one or more of the factors may be determined for a population of imaged objects. mean or variance or standard deviation. As described in more detail below, such information can be indicative of the nature of the microorganisms in the suspension, and can be used to select an appropriate calibration curve for use in determining the concentration of intact microorganisms therein. Furthermore, such information can provide information on the staining efficiency of microorganisms in suspension and can be used to determine the proportion of microorganisms whose fluorescence intensity is below the detection limit.

Images can optionally be subjected to a background subtraction or normalization step as an initial step, ie, prior to any subsequent image analysis steps described herein. This can be done using any convenient known standard method (eg rolling ball subtraction).

Image analysis values may be determined after thresholding has been performed. In other words, a threshold can be set to determine whether an object has been detected. Thresholding can be performed to set a lower limit on the intensity of the signal obtained for the image of the suspension, below which objects are not considered. In the case of the method of the present invention, thresholding allows objects with low fluorescence intensity at the emission wavelength (ie, objects that are not strongly stained by the stain) to be discarded in any future analysis. Thresholds can be set at at least one or more levels, and objects can be counted at different thresholds.

In some embodiments, global thresholding may be performed, ie, a single threshold may be set for the entire image (or set of images). However, in alternative embodiments, local thresholding may be performed (eg, if the illumination and/or background signal is not uniform across the image). Local thresholding evaluates the threshold for a given pixel based on the grayscale information of neighboring pixels.

Additionally, other image analysis operations may be performed according to techniques known in the art, such as to convert the image to grayscale (wherein the fluorescence intensity may be read as a grayscale level) prior to determining the image analysis value, and/or Subtract the background (eg using the rolling ball method), etc.

Suspensions can be characterized based on information obtained from imaging, eg, whether the microorganisms are aggregated or non-aggregated microorganisms. Advantageously, a suitable calibration curve for this process can be selected based solely on the appearance of the objects in the suspension (eg whether a certain proportion of objects detected in the suspension have a certain area and/or maximum intensity), and The identity of the microorganisms in the suspension need not be known before the concentration of intact microorganisms can be determined by the method of the present invention. Thus, a predetermined calibration curve can be selected for aggregated or non-aggregated microorganisms.

The relationship between the concentration of intact microorganisms in suspension and image analysis values may depend on a number of parameters about the microorganisms in the suspension, such as the size and morphology of the microorganisms, and/or the tendency of the microorganisms to form clusters or biofilms. Therefore, the number of objects in suspension cannot be used directly to determine the concentration of intact microorganisms in suspension, as each object may correspond to two or more microorganisms. Furthermore, in embodiments of the invention in which imaging is performed at two or more focal planes, microorganisms or clusters of microorganisms may appear in two separate images if captured at different focal planes, and thus may are detected as two separate objects. Thus, the identity of the microorganisms in the suspension may affect the relationship between the concentration of the microorganisms in the suspension and the number of objects imaged in step (e)(iv) of the method of the invention.

In the methods of the present invention, methods such as these and those previously identified in the art as affecting the accuracy of methods for determining the concentration of viable microorganisms in suspension can be overcome by the use of calibration curves (eg, by incomplete staining of intact microorganisms) the elements of.

The invention can be carried out on a series of samples (eg preparations) (or alternatively referred to as "reference suspensions") containing known concentrations of microorganisms (ie samples (suspensions) for which the concentration of microorganisms has been or has been determined by alternative methods) Steps (e)(iii) and (e)(iv) of the concentration determination method to prepare a calibration curve. Thus, the number of objects corresponding to intact microorganisms can be determined for each sample containing different concentrations of microorganisms, and thus a relationship between the number of objects and the concentration of microorganisms can be established.

The calibration curve is predetermined in the sense that the calibration curve is prepared prior to carrying out the concentration determination method of the present invention. Therefore, before determining the concentration of microorganisms in a suspension obtained from a given (ie, each) sample, a calibration curve can be prepared separately. Preferably, however, a calibration curve can be made and used to determine the concentration of intact microorganisms in multiple suspensions, or in other words, the same calibration curve can be used to determine the concentration of intact microorganisms in multiple suspensions. In other words, the method does not have to involve the generation of calibration curves; pre-made calibration curves can be used and there is no need to generate a separate calibration curve for each sample/suspension. A new or fresh calibration curve can be prepared periodically, such as daily, weekly or monthly, or can be made in batches, such as before a new batch of stain is used, and the new calibration curve can be used to determine The concentration of intact microorganisms until a new calibration curve needs to be made.

However, when carrying out the method of the present invention, a calibration curve suitable for determining the concentration of a given microorganism or microorganism type can be provided, and it may therefore be preferable to make individual microorganisms or microorganism types with different characteristics (eg different growth patterns) calibration curve. Thus, this need not necessarily be at the level of a particular microbial genus or species, but may depend, for example, on the morphology and/or growth pattern of the microorganism.

In some cases, the suitability of a calibration curve for determining the concentration of intact microorganisms in suspension may depend on the identity of the microorganisms, and the accuracy with which the calibration curve allows determination of the concentration of intact microorganisms from image analysis values will be determined. For example, it is possible that a single calibration curve generated using a specific microorganism might be suitable for determining the concentration of a range of different microorganisms (eg, microorganisms within a single family or genus), and in this way, only a single calibration curve may need to be made for use with in the method of the present invention. Alternatively, imaging data obtained from microorganisms of different families, genus, species or strains and/or different microorganisms with similar characteristics and/or morphologies can be used to make a calibration curve for this purpose, and the data obtained therefrom can be Data are combined to provide a single calibration curve.

For example, data can be collected from different species of non-aggregating Gram-negative bacteria to make a calibration curve. Therefore, the calibration curve thus produced can be used to determine the concentration of many different (suitable) microorganisms, ie there is a satisfactory (ie representative) correlation between the number of imaged objects and the concentration of microorganisms in suspension sexual microorganisms.

Alternatively, if a particular microorganism exhibits irregular or unusual properties, it may be necessary to generate a separate calibration curve for that particular microorganism in order to determine the concentration of that microorganism in suspension.

Thus, a number of different calibration curves can be provided (ie, made prior to carrying out the concentration determination method of the present invention), each suitable for determining the concentration of different selected microorganisms. Thus, for example, separate calibration curves can be provided for non-aggregating gram-negative bacteria, non-aggregating gram-positive bacteria, aggregating gram-negative bacteria, or yeast. Therefore, an appropriate calibration curve can be selected in order to determine the concentration of a specific microorganism in a sample. Thus, 2, 3, 4, 5, or 6 or more different calibration curves can be made, and once microbial imaging has been performed, an appropriate calibration curve can be selected from among them.

In a preferred embodiment of the invention, the information obtained in imaging step (e)(iv) can inform the selection of which calibration curve to use in order to determine the concentration of viable microorganisms in a microbial suspension prepared from a particular sample. One or more parameters of the object described above (i.e. the maximum intensity, mode intensity and/or area or derived value of the object as described above) may be determined for the object detected in step (e)(iv), optionally After background subtraction and/or thresholding steps, and such information can be used to select an appropriate calibration curve for the sample. Preferably, calibration curves predetermined for aggregated or non-aggregated microorganisms are used.

Factors such as the nature of the sample or suspension, the medium in which the microorganisms are resuspended, and the storage or incubation conditions of the sample and/or suspension may also affect the difference between the concentration of microorganisms in the suspension and steps (e)(iv) of this method. ) between the number of objects imaged in ), and therefore preferably the calibration curve is made under conditions similar or identical to those for imaging the suspension-stain mixture.

As mentioned above, the concentration determination method of the present invention, in the case of performing an AST assay, in particular determining the concentration of microorganisms in its inoculum, determines intact (and thus viable) microorganisms in suspensions prepared from samples is particularly useful in terms of concentration. Accordingly, the present invention provides a method for determining the antimicrobial susceptibility of microorganisms comprising preparing a suspension of microorganisms from a sample and determining the concentration of viable microorganisms in the suspension as described above, and performing an AST assay.

Advantageously, the present invention provides a method starting from a clinical sample or clinical sample culture and comprising recovering (or isolating) viable microorganisms from the clinical sample or clinical sample culture, determining that the recovered microorganisms are intact in suspension. (and thus indicates the concentration of viable) microorganisms, and optionally an inoculum is prepared from the suspension (which may include adjusting the concentration of microorganisms in the suspension or a portion or aliquot thereof). A suspension of recovered microorganisms or an inoculum prepared therefrom can be used as an inoculum for an AST microorganism test culture prepared in an AST assay.

As described further below, AST assays can be performed in any convenient or desired manner. Thus, microbial growth can be assessed (or determined) in the presence of different antimicrobial agents (eg, antibiotics) and/or multiple amounts or concentrations of antimicrobial agents (eg, antibiotics). Growth can be assessed directly or by assessing (determining) growth markers.

In general, AST assays are performed by monitoring the effect of antimicrobial agents on microbial growth. Samples containing microorganisms are used to inoculate media in a series of at least two culture vessels (ie, to establish at least two AST microorganism test cultures), each vessel containing a different concentration of antimicrobial agent, and the microorganisms are cultured for a period of time. In this manner, a series of at least two different concentrations of antimicrobial agents are tested in order to determine the amount of agent (eg, minimum inhibitory concentration (MIC)) required to prevent microbial growth. The antimicrobial susceptibility values (eg, MIC values and/or SIR values) thus obtained provide an indication of whether the microorganism is resistant or susceptible to a single antimicrobial agent.

In addition to inoculating at least two AST microbial test cultures containing different concentrations of antimicrobials, the AST assay will have positive control conditions (medium without antimicrobials) to confirm that the microorganisms are viable and capable of Growth medium provided, as well as negative control conditions (medium not inoculated with microbial cultures and without antimicrobial agents) to confirm that the growth medium was not contaminated with microorganisms not obtained from clinical samples. Thus, step (iii) of the method for determining the antimicrobial susceptibility of microorganisms in a sample will generally involve establishing suitable positive and negative control conditions in addition to at least two different growth conditions.

In some embodiments, a positive control sample can be considered to provide a first concentration of antimicrobial agent (ie, a concentration of 0M), and a second condition can be established to include only the antimicrobial agent. In such embodiments, growth in positive control conditions and conditions comprising antimicrobial agents can be assessed to determine antimicrobial susceptibility. Thus, "at least two different growth conditions in which ... each antimicrobial agent is tested at two or more different concentrations" can be considered to encompass where an antimicrobial agent is added to only a single growth condition, And the positive control condition represents an embodiment of the second concentration of antimicrobial agent.

In a preferred aspect, more than one (ie, two or more) different antimicrobial agents are tested, thereby providing two or more different values of antibiotic susceptibility (eg, MIC value and/or SIR value), one value for each different antimicrobial agent. The combination of different values (eg, different MIC and/or SIR) values provides the antimicrobial susceptibility profile of a given microorganism, i.e., to which of a set of antimicrobials the microorganism is resistant, and To which one of a group of antimicrobial agents is susceptibility. If desired, separate positive and negative control conditions can be set up for each antimicrobial agent tested, but in the case of testing multiple different antimicrobial agents, a single positive and single negative control condition is sufficient.

Microbial growth in an AST method can be assessed by any desired or suitable means, including by any means known in the art. More particularly, the growth of microorganisms can be assessed by determining the amount and/or number and/or size of microorganisms and/or microbial colonies or aggregates. As will be discussed in more detail below, in certain preferred embodiments, microbial growth is assessed (determined) by imaging or surrogate expression by visualization of the microorganism. Thus, microbial cells, which may include aggregates or clumps (clusters) of cells, or microbial colonies, can be visualized or imaged as a means of determining (or assessing or monitoring) growth. This may include, but is not limited to, counting cells or colonies, and includes by assessing microbial cells, colonies or aggregates (the term "aggregate" includes any collection of cells that are physically adjacent, such as clumps or clusters; this Can include size, area, shape, morphology, and/or number of non-clonal clumps/clusters of cells that aggregate or tangle (e.g., adjacent cells that have aggregated) as well as clonal colonies to visually assess (or determine) microbial growth amount in any way.

The parameters used to measure microbial growth can, but need not, vary depending on the identity of the microorganism and the antimicrobial agent used. Indeed, depending on the organism and the antimicrobial agent used, the morphology or growth pattern of cells may be affected, and this may vary from a "normal" or "typical" morphology or growth pattern (eg, in the absence of antimicrobial agents) circumstances) change or change. While some AST growth monitoring methods may rely on detecting such changes, such changes need not be taken into account in accordance with the present invention, and microbial growth or the amount of biomass (eg, area) can be determined without regard to morphology and/or growth pattern. Thus, the same growth monitoring method can be used regardless of the microbial cells and/or the antimicrobial agent used. Methods for performing AST determinations are further described below.

The present invention provides methods for determining the concentration of intact or viable microorganisms in suspension, and this information can be used to accurately provide specific concentrations of microbial cells in a test microbial culture. Once the concentration is determined, the concentration of microorganisms in at least a portion of the suspension can be adjusted to provide an inoculum for inoculating the test microorganism culture in step (iii). However, as mentioned above, this does not preclude additional preliminary adjustments before concentrations are determined. Thus, the concentration of microbial cells in suspension can optionally or if desired be adjusted to fall within a range suitable for use in AST assays. This conditioning may not be required in every case, i.e. the suspension can be used directly to inoculate the test microbial culture series established in step (iii) (i.e. the suspension can be used directly, i.e. without any further conditioning) . Alternatively, the suspension (or aliquot thereof) can be adjusted to a desired or predetermined concentration. Still further alternatively, the suspension can be used directly (ie, without adjustment) to inoculate a test microbial culture series, and if desired, the microbial concentration in the test microbial culture can be adjusted to a desired or predetermined concentration. Any such adjustments will be based on the concentration of viable microorganisms determined in the concentration determination method (ie, based on the concentration of microorganisms in suspension).

Accordingly, the method of the present invention may further comprise step (f), wherein the concentration of microbial cells in the suspension or a portion thereof and/or the test microbial culture is adjusted. More particularly, the concentration can be adjusted to increase or decrease the number or concentration of microbial cells. Such conditioning can be performed in the context of an AST assay, as described above, but can also be performed in any other context for any desired reason, such as conditioning an aliquot of recovered microorganisms for further analysis (e.g. genetic analysis), storage (eg, freezing), etc.

As noted above, in certain embodiments, the method may include an initial adjustment, preferably an initial dilution, prior to determining the concentration of microorganisms in the suspension. This can be considered part of the conditioning step (eg, as an initial or preliminary conditioning). Alternatively, this can be viewed as a separate initial (preliminary or blind) adjustment, which is performed independently of any adjustment steps performed after the concentration is determined. Once the concentration of microorganisms in the suspension has been determined, further adjustments can be made to the concentration of microorganisms based on the concentration of microorganisms determined in steps (e)(v) of the present invention (eg, to fall within a range suitable for use in AST determinations), if desired. range). Thus, in one embodiment, the method may comprise an additional step (f) of adjusting the concentration of microorganisms in at least a portion of the suspension after the concentration is determined in step (e). In another embodiment, the method may comprise performing an initial adjustment of the concentration of microorganisms in at least a portion of the suspension prior to determining the concentration, and then performing an adjustment to the adjusted suspension or a portion thereof after determining the concentration in step (e). further adjustment. In one embodiment, step (f) can be viewed as the step of performing such further conditioning. Advantageously, performing such an initial adjustment (eg, in the process of adjusting the concentration of microbial cells in the suspension) can reduce preparations with the desired microorganisms once the concentration of microorganisms in the suspension has been determined in steps (e)(v). The time required for a concentrated suspension such as an inoculum.

Thus, in one embodiment, the concentration of microorganisms in at least a portion of the suspension is adjusted. At least the part of the suspension in which the concentration of microorganisms is adjusted is preferably the part of the suspension obtained in step (d) that was not stained in step (e)(iii), ie it is the unstained part of the suspension.

Adjusting the concentration of at least a portion of the suspension can provide an inoculum for inoculating the test microbial culture in step (iii). Thus, for example, the concentration of microorganisms in the inoculum can be increased, for example, by culturing the sample for a period of time to allow the microbial cells to grow, or the suspension can be reduced, for example, by dilution before or during inoculation of the test microorganism culture The concentration of microorganisms in the liquid, for example, by selecting the appropriate amount (e.g. volume) to be used, by adding to solids (e.g., dried antimicrobials such as freeze-dried or vacuum-dried antibiotics) or by adding a portion of the inoculum Or aliquots were added to a volume of antibiotics and/or medium for AST testing by dilution to establish test cultures. Thus, a test microorganism culture can be inoculated with a suspension (or an aliquot thereof) or an inoculum conditioned (eg diluted) therefrom.

In one embodiment, wherein the suspension contains a higher than desired concentration of microorganisms, e.g., a concentration of microorganisms that is too high for an AST assay, the microorganism culture is diluted with an appropriate buffer or medium (e.g., liquid medium), to reduce the cell density to an appropriate level, eg, to perform an AST. In the case of an AST assay, the dilution is preferably performed using the medium used to conduct the AST assay. In one embodiment, this can be done using Muller Hinton (MH) broth. Adjusting the concentration may eg comprise dilution based on the concentration determined in step (ii) of the AST method.

In alternative embodiments, where the suspension contains a concentration of microorganisms that is too low for AST determination, the suspension can be cultured (or further cultured) for a period of time to allow the growth and number of microorganisms present therein to increase. The concentration of microbial cells present in the suspension can be monitored continuously or at a series of separate time points until the concentration of microorganisms reaches a cell density high enough for AST assays to be performed. Growth of the microbial culture at this stage can be monitored by any method described herein for monitoring growth in the AST assay itself, for example, by imaging or counting of cells or colonies, and/or can be performed after a period of growth The concentration determination method of the present invention.

Thus, in one embodiment, the present invention utilizes an inoculum (eg, a suspension or a diluted suspension) with a standard microbial concentration (eg, 0.5 McFarland units or 10 ⁸ CFU/ml) or a concentration in an area thereof for inoculation for Test cultures for AST assay. Optionally or if desired, the concentration of microbial cells present in the suspension can be adjusted (ie increased or decreased depending on the number of cells present in the sample) to obtain a suspension having a standard concentration. Alternatively, the concentration of microbial cells present in the suspension can be within a standard range, and no adjustment steps are required. In any event, the concentration of microbial cells present in the suspension is determined by the method of the present invention and can be adjusted as desired or if necessary to obtain a suspension with a standard concentration. Alternatively, the suspension can be used without adjustment, and the concentration of microbial cells in the test microbial culture can be adjusted according to the concentration of microorganisms determined in the suspension (e.g., by selecting an appropriate dilution factor to establish the test culture or suitable volume of).

AST assays typically utilize microbial cultures with set (or standard or normalized) cell densities or microbial concentrations to allow comparison of results obtained from one sample or location with results obtained elsewhere, as known The response of microorganisms to antimicrobial agents varies with the concentration of microorganisms in the sample as well as the type and concentration of the antimicrobial agent itself. Factors that affect clinical outcome (eg, dose of antimicrobial agent and treatment regimen prescribed to the patient) are based on results obtained from AST assays performed according to set standard specifications.

Results obtained in AST assays using "non-standard" (or "non-standardized") microbial cultures (an antimicrobial susceptibility profile of microorganisms, or a set of MIC and/or SIR values and/or indicating antimicrobial susceptibility Any other value of characterization) may differ from results obtained in AST assays performed according to standard practices (eg, using "standard" microbial cultures). However, if the concentration of microbial cells in the suspension or inoculum used to inoculate the AST test culture is known, it is possible to determine the antimicrobial susceptibility values obtained using a non-standard microbial culture compared to the antimicrobial susceptibility values obtained using a standard microbial culture The degree of difference between the drug sensitivity values. Thus, theoretical "standard" antimicrobial susceptibility values (eg, MIC and/or SIR values) can be calculated from antimicrobial susceptibility values obtained using non-standard microbial cultures.

The degree to which susceptibility values obtained using non-standard microbial cultures differ from "standard" MIC values may vary depending on the nature of the microorganism and antimicrobial agent, and for example for each different antimicrobial agent tested and containing different concentrations of microorganisms Microbial cultures of cells can be determined separately.

Accordingly, the present invention provides a method for determining the antimicrobial susceptibility profile of a microorganism using an inoculum comprising non-standard concentrations of microbial cells, wherein the concentration of microbial cells in a test microbial culture is measured prior to performing an AST assay (indirectly, By measuring the concentration of microbial cells in the suspension used to inoculate the test microbial culture or to prepare the inoculum), i.e., determine the concentration of microbial cells in the suspension, and based on the concentration of microbial cells in the test microbial culture thus prepared The concentration adjusts the sensitivity values (eg MIC and/or SIR values) obtained in the AST assay to obtain standard values (eg MIC and/or SIR values).

As noted above, the standard inoculum used to establish an AST test assay in the methods of the prior art is typically about 0.5 McFarland units. As mentioned above, this corresponds to about 10 ⁸ CFU/m. This is typically diluted at a dilution of 1 :200 to provide a test microbial culture containing about ^5x105 CFU/ml. However, although these standard values can be used in the methods of the present invention, and it is generally preferred that the concentration of microorganisms in the microbial test culture inoculated in the AST test is ^4.5x105 ±80% or ^5x105 ±60%, in the present invention In the method, the inoculum (e.g., the suspension and inoculum prepared therefrom) and/or the test microbial culture may comprise any defined or predetermined microbial cell concentration, provided that the microorganisms in the test microbial culture used to obtain the AST value The concentration of cells is known. Thus, in other embodiments, the concentration of microorganisms in the microbial test culture inoculated in the AST test may be in the range of 1 x 10 ⁵ ± 80% or 5 x ^{10 4} ± 80% or 5 x ^{10 4} ± 60%.

Thus, the concentration of microbial cells in suspension can be any desired or predetermined concentration suitable for establishing a microbial test culture in an AST method. Thus it may be at least 10, 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 5× ^{10 10} or 10 ¹¹ CFU/ml. The concentration of microbial cells in the suspension is preferably 10-10 ¹¹ , 10 ² -10 ¹¹ , 10 ³ -10 ¹¹ , 10 ⁴ -10 ¹¹ CFU/ml, 10 ⁵ -10 ¹¹ CFU/ml, 10 ⁶ -10 ¹¹ CFU /ml, 10 ⁷ -10 ¹¹ CFU/ml, 5x10 ⁶ -10 ¹¹ CFU/ml, 2x10 ⁶ -10 ¹¹ CFU/ml, 10 ⁶ -10 ¹¹ CFU/ml, 5x10 ⁶ -5x10 ¹⁰ CFU/ml, 2x10 ⁶ - 5x10 ¹⁰ CFU/ml, or 10 ⁶ -5x10 ¹⁰ CFU/ml.

The statistical reliability of AST determinations using an inoculum with a low concentration of microorganisms may be less than that of an embodiment in which the inoculum contains a higher concentration of microorganisms. Thus, in certain embodiments, if a particularly low concentration of microorganisms is determined in the suspension, it may be desirable or advantageous not to proceed with the AST assay at this stage. Thus, in certain embodiments, when the concentration of microorganisms in the suspension is below ^1x103 CFU/ml, or more preferably below ^1x104 , ^1x105 or ^1x106 CFU/ml, the AST assay can be performed without the suspension (ie, no AST method beyond step (ii) is performed). Optionally, the concentration of microorganisms in the suspension can be allowed to increase (e.g., after a period of incubation) before repeating the concentration determination method, and if at this later stage the suspension contains a sufficiently high concentration of microorganisms, then an AST can be performed. Determination.

The AST method of the present invention, which allows the use of non-standard concentrations for AST testing, is of particular utility if the concentration of microbial cells in the suspension is lower than the standard concentration, as it can bypass the need to incubate the suspension for a period of time to allow the The microbial cell concentration in the suspension is required to increase, for example, to a level higher than the standard concentration.

The AST methods presented herein can be viewed as methods for determining the "standard" antimicrobial susceptibility profile of microorganisms by adjusting the sensitivity (eg, MIC and/or SIR) values obtained from AST assays using non-standard microbial cultures . Viewed in another way, this provides a theoretical method to adjust the concentration of microbial cells used to inoculate the test cultures used in the AST assay, thereby calculating the antimicrobial susceptibility of the microorganisms.

Although non-standard samples can be used to inoculate test cultures for use in the present invention, in alternative embodiments, the present invention provides methods for physically adjusting the concentration of microbial cells present in suspensions and/or test microbial cultures, A standard AST assay can be performed such that the concentration of microbial cells in the test microbial culture corresponds to a standard or normalized concentration (eg, about ^5x105 CFU/ml).

The suspension or the inoculum prepared therefrom is used to inoculate the test microorganism culture. As mentioned above, in the stage of establishing the test microorganism culture (step (iii) of the AST method), the suspension can be added to the medium, ie the suspension can be diluted or further diluted. Thus, the culture of the test microorganism can be adjusted at this point to contain any desired or predetermined concentration. Thus, the test microbial culture will contain at least 10, 10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ or 10 ⁹ CFU/ml, preferably 10 ² -10 ⁸ , 10 ³ - Initial concentration of microbial cells of 10 ⁷ or 10 ⁴ -10 ⁶ CFU/ml. As described above, therefore, test microbial cultures can be established to final concentrations of ^5x104 ±80%, ^1x104 ±80%, ^4x105 ±80%, ^4.5x105 ±80% or ^5x105 ±80%.

It should be noted, however, that the conditions constituting a "standard" sample may vary depending on the identity of the microorganism, ie the concentration of microbial cells present in the suspension may depend on the identity of the microorganism. The concentration of microbial cells in the suspension is preferably 10-10 ¹¹ , 10-10 ¹⁰ , 10-10 ⁹ , 10 ² -10 ⁹ , 10 ³ -10 ⁹ , 10 ⁴ -10 ⁹ CFU/ml, 10 ⁵ -10 ⁹ CFU/ml, ^{106-109 CFU} /ml, ^{107-109 CFU} /ml.

There are well-established and prescribed conditions for AST assays and can be followed so that comparable results that are comparable or comparable to tests performed in other laboratories can be readily obtained.

For example, this can involve the use of defined media and culture conditions. In certain embodiments, the medium used for microbial culture can be a liquid medium, ie, the medium can be a liquid.

In certain embodiments, it may be advantageous or desirable to establish test microbial cultures in parallel with different media for growth of different microorganisms. This may be useful, for example, if the identity of the microorganisms in the sample (and thus the suspension) is unknown, or if their growth patterns or requirements have not been fully characterized. Thus, for example, parallel microbial test cultures in the AST method can be established with or without fastidious supplements in the growth medium, or in other words, in fastidious or non-fastidious mediums Parallel test microbial cultures in fastidious or non-fastidious media. Fastening media are well known in the art, and both pre-prepared fastidious media and fastidious supplements are widely commercially available. Fasting supplements include, for example, lysed blood preparations (eg, lysed horse blood), serum, multivitamins and/or minerals, cofactors, and the like, such as beta-nicotinamide. Conveniently, the fastidious supplement can be added to the medium as part of a dilution regimen. Furthermore, whether to use a fastidious medium or a fastidious supplement may depend on the concentration of microbial cells determined for the suspension. For example, if the concentration is low, eg, the microbial cells in suspension are less than ^2x106 CFU/ml, the use of a microbial test culture containing a fastidious medium/supplement can be omitted from the AST method.

In certain embodiments, it is also advantageous to establish test microbial cultures in parallel with different media optimized for testing susceptibility to a particular antimicrobial agent. Test microbial cultures may contain additives necessary for specific antibiotics. For example, polysorbate 80 may be included, and/or may provide increased calcium concentrations in certain test microbial cultures.

Microorganisms can be grown in the presence of various antimicrobial agents to determine their susceptibility to a given antimicrobial agent. Antimicrobial agents can be selected based on the identity of the microorganism (if known), and preferably also based on the nature of any inherited antimicrobial resistance markers identified within the microorganism. The antimicrobial agent and amount used may also be selected according to current clinical practice, such as which antimicrobial agent is currently used in practice to treat the identified microorganism, so that the microorganisms can be assessed against the currently accepted or recognized selected antimicrobial agents. Sensitivity to drug therapy.

Accordingly, antimicrobial agents may be selected based on antimicrobial agents known to be effective against the identified microorganism or antimicrobial agents currently used in practice to treat the microorganism, and excluding antimicrobial agents that may be expected to have, based on the presence of resistance markers, Any agent of resistance, or despite the presence of a marker of resistance, such an agent may be included and the amount used may be selected to allow determination of the amount or concentration of potentially effective antimicrobial agent. Antimicrobial agents are added to the medium at a range of final concentrations or amounts. In one embodiment of the present invention, dilution of the antimicrobial agent may be performed. In a preferred form of the invention, a predetermined amount of antimicrobial agent, which upon dissolution yields a predetermined concentration, is pre-deposited in wells to which a medium containing microorganisms is added prior to AST. The pre-deposited antimicrobial agent is preferably a dry, eg freeze-dried or vacuum-dried formulation.

The step of growing or culturing the suspension/microorganisms therefrom in an AST assay can be performed by any known or convenient means. Solid phase or liquid phase culture can be used.

Thus, for example, in a preferred embodiment, microorganisms can be cultured on or in a plate or other solid medium containing the antimicrobial agent or in a vessel (eg, wells of a plate) containing a liquid medium containing the antimicrobial agent, And microbial growth can be determined by microbial visualization (eg, imaging) (eg, plate imaging, etc.). Therefore, cultures are directly visualized or imaged as a means of monitoring or assessing growth. Therefore, in a preferred embodiment, the culture is directly analyzed to monitor/assess growth. For example, cultures can be grown in wells of a plate or compartments of a carrier substrate, and the wells/compartments can be imaged.

Alternatively, samples (or aliquots) can be removed (or taken) from the AST test culture at intervals or at different time points, and the removed samples (aliquots) can be analyzed for microbial growth. This can be accomplished by any means, including, for example, by molecular tests, such as nucleic acid-based tests. Thus, detection probes and/or primers that bind to microbial cells or to components released or isolated from microbial cells can be used. This can include, for example, nucleic acid probes or primers that can hybridize to microbial DNA. In other embodiments, as described in more detail below, the microbial cells can be detected directly, eg, by staining.

Each antimicrobial agent can be used in at least two concentrations, with the exception of a positive control that allows growth of microorganisms in the absence of any antimicrobial agent and at least one negative control that is incubated without addition of aliquots . For example, 2, 3, 4, 5, 6, 7, or 8 or more concentrations of antimicrobial agents are used. The concentrations used in the dilution series may vary by a factor of two between individual concentrations.

The term antimicrobial agent includes any agent that kills microorganisms or inhibits their growth. Antimicrobial agents of the present invention may include, inter alia, antibiotics and antifungal agents. Antimicrobial agents can be microbicidal or microbiostatic. A number of different classes of antibiotics are known, including those that are active against fungi or groups of fungi in particular, and any or all of these antibiotics may be used. Antibiotics may include beta-lactam antibiotics, cephalosporins, polymyxins, rifamycins, lipiarmycins, quinolones, sulfonamides, macrolides, lincosamides, tetracyclines, aminoglycosides , glycopeptides, cyclic lipopeptides, glycocyclines, oxazolidinones, lephamycins or carbapenems. Preferred antifungal agents of the present invention may include polyenes, imidazoles, triazoles and thiazoles, allylamines or echinocandins. Antimicrobial agents are constantly being developed, and it should be understood that it is also possible to analyze future antimicrobial agents using the present invention.

Preferably, the at least one test microbial culture comprises a fastidious medium. More preferably, the at least two test microbial cultures (e.g., at least two different growth conditions comprising different concentrations of the same antimicrobial agent) may comprise a fastidious medium such that the microorganisms are resistant to a particular antimicrobial agent under the fastidious growth conditions. Has antimicrobial susceptibility.

Antimicrobial susceptibility can be determined by culturing microorganisms from suspension and analyzing AST cultures over a period of time.

AST cultures can be analyzed at multiple time points to monitor microbial growth. For example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 after the start of culture , 22, 23, or 24 hour time points to analyze cultures. Cultures can be analyzed immediately after initiation of culture, where t=0. Cultures can also be analyzed beyond 24 hours after the start of the culture. Typically, cultures can be analyzed at 0, 1, 2, 3, 4, 6 and 24 hours after initiation of culture. However, the results obtained using this method suggest that shorter incubation times may be sufficient to detect differential growth of microorganisms, such as 4 hours. Therefore, shorter total incubation times of up to 8, 7, 6, 5, 4, 3 or 2 hours, eg every hour or every 2 hours or 90 minutes, can also be used for analysis. As mentioned above, cultures are typically analyzed at two or more time points, eg, at two or more time points in culture for up to 4, 5 or 6 hours. In certain embodiments, AST cultures can be analyzed at more frequent time points. Cultures can be analyzed at t=0, and then at 1, 2, 3, 4, 5, 10, 15, 20, 25 or 30 minute intervals. Thus, the overall incubation time required when using such short analysis intervals can also be advantageously reduced, so shorter incubation times as long as 10, 15, 20, 25, 30 or 60 minutes can be used.

Monitoring or assessment of microbial growth in an AST assay can be performed either continuously or over a period of time (e.g., up to 10, 15, 20, 25, or 30 minutes or up to 1, 2, 3, 4, 5, 6, Growth was monitored at intervals of 7 or 8 hours, or by comparing the amount of microbial cellular material at the beginning (t0) of an AST growth culture (test microorganism culture) with subsequent time points (e.g., up to 10, 15, 20, 25, or 30 minutes or The amount of microbial cellular material up to 1, 2, 3, 4, 5, 6, 7 or 8 hours) was compared, ie the growth that occurred during the interval. Alternatively, the amount of microbial cellular material can be determined at two or more different time points (eg, measuring 1, 2, 3, 4, 5, 10, 15, 20, 25 or 30 minutes or 1, 2, The first time point after 3 or 4 hours and measuring 1, 2, 3, 4, 5, 10, 15, 20, 25 or 30 minutes or 1, 2, 3, 4, 5, 6 after the first time point or a second time point of 7 hours, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55 minutes after the start of the culture or 2, 3, 4, 5, 6, 7 or 8 hours at a second time point), so that the amount of growth can be determined. In a preferred embodiment, the degree of growth of the microorganism can be determined at more than one time point, ie at least two time points.

In another embodiment, the test microbial culture grown in the presence of the antimicrobial agent is assessed at only one time point, eg, at 1, 2, 3, 4, 5, 6, 7, or 8 hours, and in the absence of Growth of test microbial cultures (eg, positive controls) grown in the presence of antibiotics. Monitoring growth at a time point (or two or more time points) after the start of an AST growth culture can be beneficial for more accurate results by avoiding measuring growth during the lag phase of microbial growth due to different conditions during this time Any differences in microbial growth below will be small and difficult to detect. A first measurement according to this method can be taken after 30 minutes or 1, 2, 3 or 4 hours and a second measurement can be taken 1, 2, 3, 4, 5, 6, 7 or 8 hours after the first time point Measurement).

However, it is clear that for certain microorganisms, such as certain anaerobic bacteria, mycobacteria or fungi, microbial growth may be slower and therefore AST measurements may take longer. Thus, according to certain embodiments of the present invention, it may be necessary or desirable to perform an AST assay by measuring microbial growth for 8, 9, 10, 11, or 12 hours or more (eg, 12, 18, or 24 hours). Appropriate measurements may be taken at one or more time points accordingly.

In a preferred embodiment, growth can be measured under at least two growth conditions (eg, each growth condition) relative to the initial number (amount or concentration) of microbial cells under each growth condition.

Cultivation of the test microorganism culture can be carried out at any temperature that promotes the growth of the microorganism, for example between about 20°C and 40°C, or between 20°C and 37°C, preferably between about 25°C and 37°C, more preferably about 30°C to 37°C or between 30 and 35°C. In one embodiment, AST cultures can be grown at about 35°C.

Many methods for monitoring or assessing microbial growth are known and used in AST assays, including, for example, turbidity measurements, colorimetric assays, light detection, light scattering, pH measurements, spectroscopic measurements, fluorescence detection, measurement of antibiotics or microbial degradation product, measure nucleic acid content or measure the production of gas (eg CO ₂ ). Any of these can be used. However, according to a preferred embodiment of the present invention, growth can be detected and assessed by imaging methods by determining or assessing the number and/or amount and/or size and/or area of microbial cells. As mentioned above, microbial cells can include cells in colonies and/or aggregates. This can be accomplished by assessing or determining the number or amount of microorganisms present before and/or after growth in the presence of an antimicrobial agent using any method known to measure or detect microorganisms. Such determination may involve determining the number and/or size of microbial cells, aggregates and/or colonies. Again, techniques for this purpose are known and available. Thus, growth can be measured by monitoring the number and/or amount and/or size of microorganisms and/or microbial cells and/or colonies and/or aggregates over time. This can be measured directly or indirectly. The number or amount of microorganisms can be directly measured by blood counts, flow cytometry or automated microscopy. The microorganisms can be immobilized and/or permeabilized prior to detection. Alternatively, microorganisms can be detected under in vivo conditions.

An AST assay method for monitoring bacterial cell counts by using flow cytometry is described in Broeren et al., 2013, Clin. Microbiol. Infect. 19.286-291. Price et al., 2014, J.Microbiol.Met. 98, 50-58 and Metzger et al., 2014. J.Microbiol.Met. B2) describes a method for performing an AST assay in which cells are grown and enumerated by automated microscopy in a multi-channel fluidic cartridge. In these methods, bacteria are immobilized and grown on a surface, and the viability and/or growth of individual bacteria and/or colonies is assessed by imaging the surface at two or more time points (including measuring the growth). Such a method can be used according to the present invention. Fredborg et al, J Clin Microbiol. 2013, 51(7):2047-53 and Unisensor (US 8780181) describe other known methods in which brightfield microscopy is used by taking a series of stacked images (object planes) of the solution ) and count the bacteria present in the sample to image the bacteria in solution.

Any method described herein or known in the art based on the use of imaging to monitor microbial growth can be used in the AST step of any of the methods disclosed herein for determining AST (step (iv) of the AST method above). However, in certain embodiments, the microbial growth determination step in the AST method of the AST method (ie, step (iv) of assessing the extent of microbial growth) does not rely on counting individual cells or monitoring the growth of individual cells or colonies (e.g. , monitoring the increase in individual cell or colony size according to the method of Accelerate Diagnostics Inc). Accordingly, the methods disclosed herein are not limited to (and in certain embodiments do not involve) imaging AST cultures or AST culture samples using fixed positions. Furthermore, bulk growth of cells in the AST culture is preferably monitored, eg by imaging bulk cells within the field of view. The amount (eg, area) of microbial cellular material (biomass) in the field of view can be determined by imaging. Cells/microbial biomass can be detected directly (eg by microscope or camera etc.), eg using brightfield microscopy, or microbial cells can be stained for detection eg by feeding AST cultures after a predetermined or desired time of growth Or add stains to culture samples. However, in other methods, individual cells can be counted, or the growth of individual cells or colonies can be monitored. Accordingly, methods other than those specifically described and demonstrated herein can be used to determine or assess microbial growth in AST test cultures, and the methods disclosed herein for preparing microbial suspensions and/or determining microbial concentrations therein can be used in other AST methods.

Thus, in step (iv) of assessing growth in a microbial test culture, this is preferably done by imaging the test culture over a large (significant or substantial) portion of the culture available for imaging. Furthermore, in step (iv), imaging can be accomplished without preselecting a population or portion of the test culture for imaging. Time-lapse images of liquid (broth) cultures can be produced.

In another specific embodiment, the microbial cells can be localized to a detection site or surface for imaging without immobilizing them or driving or actively transporting them to a surface (eg, without applying a force, such as electrophoresis) AST cultures were directly imaged or visualized.

In such imaging methods, algorithms can be applied to determine the value of microbial growth from the images according to methods and principles well known in the art. Thus, statistical methods can be applied to microbial cell images based on the number, size and/or area of microbial cellular material/biomass in the image (eg, amount of all microbial cellular material in the image/field of view, eg, total cellular material imaged). Based on the identity of the microorganisms and antimicrobial agents present in the culture, algorithms can be written to account for different growth patterns and/or morphologies. Exemplary image analysis algorithms for measuring the amount of microbial biomass and thus microbial growth in a sample are described in co-pending application WO 2017/216312, which combine thresholding and texture filtering, and such methods can be used for The growth of microorganisms in the AST method of the present invention is assessed. This enumeration or imaging method allows for digital phenotyping of microorganisms in AST assays. The data obtained indicate that this digital phenotyping assay provides MIC values similar to those of reference techniques such as microbroth dilution.

A particular advantage of using such a method is that antimicrobial susceptibility testing can be performed on test microbial cultures containing a wide range of concentrations or amounts of microorganisms, and it is not necessary to use standardized microbial titers prior to antimicrobial susceptibility testing. . A useful feature of the present invention is the ability to use different concentrations of microorganisms. A test microbial culture or sample comprising at least 10 ³ CFU/ml can be used in the method of the invention, for example a culture or sample comprising at least 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ can be used or 10 ¹¹ CFU/ml samples (AST test samples). Alternatively, a test microbial culture or sample containing less than 10 ³ CFU/ml, eg at least 10 ² CFU/ml, can be used. In the methods of the present invention, test microbial cultures or samples containing less than 10 ² CFU/ml can also be used.

DyeCycle^TM对微生物内的DNA进行染色。其他DNA染色剂是周知的且可用。实际上，在本领域中可用于细菌染色的染色剂的数目是巨大的，并且大量这样的染色剂已有记录(包括在标准参考文献中)，并且是市售的，例如来自Life Technologies。通过染色直接标记微生物是容易进行、方便且具有成本效益的，因此代表了优选的实施方案。Although brightfield imaging represents one format for determining the concentration of microbial cells in a test microbial culture, in one embodiment of the invention, the Microorganisms are detected either by markers that stain (ie, stains or dyes) by methods that use intrinsic properties of the microorganisms (eg, phase contrast) or any other method known in the art for quantifying the number of bacteria in a sample. Suitable stains may include colored or fluorescent dyes, such as Gram stain or other stains of peptidoglycan or DNA staining, as a means of visualizing microorganisms. In a specific embodiment of the invention, it is possible to use

DyeCycle ^™ stains DNA within microorganisms. Other DNA stains are well known and available. Indeed, the number of stains available in the art for bacterial staining is enormous, and a large number of such stains have been documented (including in standard references) and are commercially available, eg, from Life Technologies. Direct labelling of microorganisms by staining is easy, convenient and cost-effective and therefore represents a preferred embodiment.

Thus, for example, microorganisms can be grown in wells of a microtiter plate (ie, each test microbial culture can be in a well of the plate) for AST assays, and stains or dyes can be added at the end of the growth period And plate wells can be imaged, and the number or amount of microorganisms or microbial cellular material can be assessed by determining the number and/or size of microbial cells, aggregates or colonies (eg, by counting or imaging). Alternatively, microbes can be counted using a flow cytometer or similar type of instrument (eg Aquila 400 instrument from Q-linea AB (Sweden)) as described in US Pat. No. 10112194.

Algorithms for image analysis to be able to analyze the image and derive or obtain values for microbial biomass, etc. are well known and available in the art. As mentioned above, one such image analysis technique is described in WO 2017/216312 and this represents the preferred way of assessing and determining microbial growth in AST testing.

Other algorithms can be used to derive antimicrobial susceptibility values (eg, MIC and/or SIR values) of microorganisms in a sample to one or more antibiotics. In this regard, while the identification of microorganisms can aid in the establishment of an AST test, it is not a prerequisite for the method and knowledge of the microorganism ID is not required to perform or establish the method. Thus, in terms of speed of test results, the AST method can be initiated when the identity (ID) of the microorganisms in the sample is unknown, but the ID can be used for interpretation of the results, for example, when imaging AST microorganism test cultures, and/or When analyzing the results of imaging. Antimicrobial susceptibility values (eg, MIC values) can be obtained without microbial ID, but ID information is important for determining or interpreting SIR (susceptibility/intermediate/resistance) information for microorganisms. Data processing techniques for deriving or obtaining MIC and/or SIR information from growth data obtained from imaging analysis are well known and available to those skilled in the art.

In an alternative embodiment, microorganisms can be specifically labeled by biological features in or on the microorganisms. A "biological feature" can be, for example, a molecule in or on a microorganism, such as a protein or other biomolecule expressed or localized on the cell surface. For example, labels (eg, colored or fluorescent labels) can be conjugated to proteins or other affinity binding molecules that specifically bind a particular biological feature. In one embodiment, the protein may be a lectin, an avidin, or an antibody, or an antibody fragment. Microorganisms labeled in this way can be detected, eg, enumerated, as previously described.

In another embodiment, proximity probes can be used to detect specific biological features in or on microorganisms.

In another alternative embodiment of the present invention, padlock probes and RCA-based amplified single molecule detection (ASMD) methods can be used to detect and enumerate microorganisms in test microbial cultures. Such methods enable detection and enumeration of individual microbial cells. Thus, microorganisms can be detected by the binding of padlock probes, and the number of microorganisms can be indirectly measured by the amplification signal generated by the RCA of the circularized padlock probes. Each RCA product (spot) may represent a single microorganism. Microorganisms can be lysed and padlock probes designed to hybridize to one or more nucleotide sequences of the microorganisms can be used. This may include the step of isolating the DNA, and preferably selectively isolating or enriching the microbial DNA. Since testing microbial cultures in AST assays is often less complex than in initial samples, simplified protocols can be used to isolate or enrich microbial DNA, such as involving filtration to isolate microorganisms and microbial cell lysis or simply to perform microbial cell lysis directly .

Alternatively, affinity binding molecules that bind to one or more molecules present on or within a lysed microorganism can be used, such as affinity probes provided with nucleic acid labels or tags that can hybridize to padlock probes , ie similar to the immune RCA (immuneRCA) detection procedure. Similarly, proximity probes can be used to bind to targets in or on the microorganism, and the nucleic acid domains of the proximity probes can be used to template padlock probe ligation, and also optionally prime its amplification by RCA. Procedures for this purpose are well known and described in the literature. Rolling circle amplification (Circle -to-circle amplification, C2CA) can be used for signal amplification. The number of microorganisms in a sample can thus be assessed by counting the number of spots, which can be labeled, as above for "spots" in a sample fluorescently labeled. Thus, this provides another convenient means of obtaining digital phenotypic sensitivity readings.

In general, it is advantageous to perform an AST assay that is pure for the microbial culture to be tested, ie, a single microorganism. However, this is not a required feature, and AST assays can be performed using visualization or imaging-based microbial detection methods, such as those offered by Accelerate Diagnostics, which use imaging of bacteria on surfaces rather than in solution, or actually in fluidic systems Methods of detecting labeled microorganisms, such as automated microscopy-based methods as discussed above in Price et al., 2014, J.Microbiol.Met. 98, 50-58 and Metzger et al., 2014. J.Microbiol.Met. system of fluid cartridges. Any cell-by-cell detection or shape recognition and/or identification method can be used for AST determination of samples containing more than one microorganism. It is further known that the same antibiotic may have different effects on different microorganisms, and therefore, the appearance of organisms after treatment with a particular antibiotic can be used to identify and AST assay each microorganism in a co-culture.

Conveniently, the method of the present invention may be automated. Any of the various steps may be automated, preferably any or all of steps (a) to (e). Many of the specific or preferred steps discussed above are inherently highly automated, such as contacting an aliquot with a stain and/or diluting an aliquot and/or aliquot of a suspension in the concentration determination method of the present invention Specimen/stain mixture imaging, as well as AST determination and recovery of microorganisms from samples. Automated culture methods, including blood culture methods, have been developed and can be combined with automated concentration determinations and/or AST assays, eg, used in accordance with the present invention. Automation will bring the advantages of speed and ease of operation, as well as multiplexing capabilities, which are important in clinical laboratory settings and especially in the diagnosis of sepsis.

The present invention and/or the methods disclosed herein will now be described in more detail in the following examples with reference to the following figures.

In the picture,

Figure 1 shows that there is a linear relationship between sample dilution and calculated microbial concentration using the method of the present invention. The calculation of Enterococcus faecalis concentration is exemplified.

Figure 2 shows the effect of varying the ethanol concentration used for cell fixation in the detection of microorganisms. The two strains of P. mirabilis detected are shown (20170927crl1 is the top line).

Figure 3 shows the results of the analysis of Proteus mirabilis as described in Example 2. The vertical dashed line is the lower bound; the vertical solid line is the lower 2.5% quantile; the normal distribution of the results is also shown. Each point corresponds to each data point (CFU/ml calculated from plating and colony count). The numerical data represented is displayed below the graph.

Figures 4-12 show the same data as Figure 3, but for Klebsiella pneumoniae (Figure 4), Haemophilus influenzae (Figure 5), Escherichia coli ( Figure 6), Enterobacter cloacae (Figure 7), Acinetobacter baumanii (Figure 8), Streptococcus pneumoniae (Figure 9), Pseudomonas aeruginosa ) ( FIG. 10 ), Staphylococcus epidermidis ( FIG. 11 ) and Staphylococcus aureus ( FIG. 12 ).

Figure 13 shows the concentrations of the range of microbial concentrations present in different positive blood culture bottles compared to the steps for concentration determination and concentration adjustment, and the resulting microbial concentrations if a fixed dilution factor were applied to the aliquots . BCF sample microbial concentrations are shown as solid squares, fixed dilution sample concentrations are shown as open squares, and samples for which microbial concentrations have been determined/adjusted are shown as circles. A dashed line of ^5x105 CFU/ml ± 60% is shown (EUCAST, CLSI and ICO criteria).

Figure 14 shows microbial biomass as a function of time during incubation of clinical Klebsiella pneumoniae isolates under different conditions. Automated microscopy images were acquired at t=30, 90, 150, 210, 270 and 330 minutes. Figure 14A - Microbial biomass in the presence of trimethoprim/sulfamethoxazole dilution series. Figure 14B - Microbial biomass in the presence of piperacillin/tazobactam dilution series. Antibiotic concentrations are measured in mg/l.

Example

Example 1 - Microorganism Concentration Determination

Material preparation

Blood Lysis Buffer:

A 0.45% Brij-O10 (Sigma-Aldrich, P6136) solution was prepared in PBS pH 7.5 (10x PBS (Sigma-Aldrich, _P7059 ) diluted to 1x with ddH2O).

Proteinase K:

Proteinase K (Merck, 539480-1GM) was dissolved in 50 mM Tris-HCl, pH 8, at a concentration of 2.1 mg/ml to give a stock proteinase K solution.

SYTO BC:

5 mM SYTO BC (Thermo Fisher Scientific, S34855) was added to 1x PBS to obtain a 20 μM SYTO BC stock.

Concentration Determination Protocol

Mix 1 ml of lysis buffer with 50 μl of proteinase K stock. The resulting lysis buffer/proteinase K mixture was added to 500 μl bacterial sample and mixed. The mixture was incubated at 35°C for 7 minutes and then filtered through a 50 mm diameter filter with a pore size of 0.2 μM at a rate of 4 ml/min.

Isolated bacteria were washed with 2 ml CAMBH (Thermo Fisher Scientific, T3462) and then resuspended by backwashing through the filter with 2.5 ml CAMBH at 4 ml/min. The resuspension is then mixed.

20 μl of the resuspension was then mixed with 0-20 μl of 70% ethanol; the volume of the resuspension/ethanol mixture was made up to 40 μl with ddH ₂ O such that the resulting ethanol concentration was 0-35%. The mixture was incubated at 35°C for 5 minutes. 60 μl of PBS was then added, and 20 μl of the resulting dilution mixture was used to make a 10-fold serial dilution.

A 15 μl aliquot of each diluted sample was mixed with 15 μl S YTO BC stock solution and the stain-sample mixture was incubated for 5 min at 35°C. The stain-sample mixture was then transferred to the plate and read in an Etulama plate reader.

During reading, the SYTO BC emission peak at 509 nm was detected using an emission filter of 502-561 nm, thereby obtaining 50 images for microorganisms in suspension in each well 30 μm apart in the direction of the optical axis. The acquired images were thresholded and analyzed to determine the size, fluorescence intensity, and optional morphology of each object corresponding to intact microorganisms to obtain image analysis values for each aliquot. The characteristics of the microorganisms in the sample are used to select a predetermined calibration curve for use in concentration determination steps (eg, to determine whether the sample is an aggregated or non-aggregated microorganism). A diluted aliquot with image analysis values within the predetermined calibration curve range was identified. The concentration of intact microorganisms in the sample is determined by comparing the image analysis values of selected diluted aliquots to a predetermined calibration curve.

Make a calibration curve

Imaging data was collected as described above for many different microorganisms at different known concentrations and using different concentrations of ethanol as fixative, and the relationship between the number of objects counted and the concentration of intact microorganisms was plotted on a graph. When a given concentration of ethanol was used as the fixative, there was a linear relationship between the number of objects counted for most microbial species and the concentration of intact microorganisms, as shown for E. faecalis in Figure 1 (using 35% ethanol as the fixative ).

The optimal concentration of ethanol as a fixative was determined for a variety of microbial species and strains. For many of the species and strains tested, an optimal ethanol concentration of about 35% was determined that maximizes microbial staining, improving detection and increasing the accuracy of concentration determination. Figure 2 shows an exemplary graph demonstrating the concentration determination of two strains of Proteus mirabilis using different concentrations of ethanol as fixative. For each ethanol concentration used, the same concentration of bacteria was present in the sample. As shown, 30-35% ethanol concentrations provided the best detection for both strains.

Example 2 - Analysis of Various Bacterial Species

Samples including the following species were analyzed according to the method of Example 1: Proteus (Figure 3), Klebsiella pneumoniae (Figure 4), Haemophilus influenzae (Figure 5), Escherichia coli (Figure 6), Enterobacter cloacae Bacillus (Figure 7), Acinetobacter baumannii (Figure 8), Streptococcus pneumoniae (Figure 9), Pseudomonas aeruginosa (Figure 10), Staphylococcus epidermidis (Figure 11) and Staphylococcus aureus (Figure 12) .

As shown, between 10 and 36 samples including each species were analyzed (see "N" values). The bacterial concentration of each sample was determined by plating followed by CFU counts. The data were fitted with a normal distribution and two values were labeled for each dataset: the lower 2.5th quantile and the bacterial concentration at the lower limit of detection using the method of the invention (corresponding to 1.5x10 ⁶ CFU/ml ).

To ensure the accuracy of the method of the invention, preferably, for each species, there is at least 1 order of magnitude between the limit of accurate concentration determination and the concentration of the lower 2.5% quantile of the sample. As shown in Figures 3-12, this was the case for all species except P. aeruginosa, S. epidermidis, and S. aureus. For P. aeruginosa and S. epidermidis, the difference is slightly less than 1 order of magnitude; although this is not optimal, it is still expected that the methods of the present invention are highly accurate in measuring concentrations of these species. For S. aureus, the limit for accurate concentration determination is the 4th quantile. This is due to aggregation of S. aureus, and it is believed that isolation of S. aureus clusters (eg by using detergents or suitable algorithms) will overcome this difficulty.

Example 3 - Preparation of inoculum from positive blood culture flasks

We studied a variety of different Gram-positive microbial species (Strep Concentrations of microorganisms in positive blood culture flasks for Staphylococcus capitis, Staphylococcus hominis, Enterococcus faecalis, Listeria monocytogenes and Listera Grayi) variability. Determine the viable cell count for each positive blood culture flask and determine a fixed dilution factor based on the average concentration of microorganisms in each blood culture flask. An aliquot of each positive blood culture bottle was diluted with this fixed dilution factor.

Microbial suspensions were prepared from each positive blood culture flask and viable cell counts were determined for each resuspension. The microbial concentration of the resuspensions obtained from each positive blood culture flask was also determined by the method outlined in Example 1 above, except that the microorganisms were resuspended in 2.8 ml of CAMBH. An inoculum was prepared for each sample based on the determined microbial concentration. Calculate the actual concentration of viable cells provided in each inoculum by the following formula:

Only 28% of the positive blood culture samples diluted with the fixed dilution factor had viable cell concentrations within the standard ^5x105 CFU/ml ± 60% range for AST, whereas 87% of the sample prepared inoculum (based on the concentration determination method) was found. adjustment) within this range. The results are shown in Table 1 and FIG. 13 .

Table 1 - Microorganism concentrations in positive blood culture bottles and diluted aliquots

Example 4 - Isolation, concentration determination of adulterated positive blood culture flasks using clinical isolates of Klebsiella pneumoniae and antibiotic susceptibility

Material preparation

Preparation of positive blood culture samples

Clinical isolates grown on agar plates were individually suspended in PBS and adjusted to 0.5 McFarland. A 1:100 dilution of this was added to a blood culture bottle (BD Bactec Plus Aerob) along with 9 ml of blood from a healthy donor and incubated overnight in a blood culture cabinet. In the morning, after BCF became positive, a 500 μl aliquot of positive BCF was used for subsequent analysis.

Sample Preparation:

500 μl of positive BCF was added to the consumable to allow for automated sample preparation and concentration adjustment, and the concentration of microorganisms was determined in an automated system as described in Example 1, except that the microorganisms were resuspended in 2.8 ml of CAMBH. The operation of such a system using this consumable is described in more detail in our co-pending application GB 1806505.2. The concentration determinations were compared to a predetermined standard curve, and the concentration of microorganisms in the recovered suspension was automatically adjusted to the desired concentration (5x10^5 CFU/ml). For this experiment, aliquots of concentration-adjusted bacteria were plated on agar plates to determine viable cell counts to provide a control measurement for this method.

AST

Concentration-adjusted samples in CAMBH were added to 336-well AST discs prefilled with various concentrations of dry antibiotics using an automatic pipette through the central injection port.

Each well contained 20 μl of sample and was incubated at 35°C. An initial reading (reading 0) was taken after 30 minutes. Imaging by automated microscopy followed by taking readings every hour (readings 1-6) up to a total AST time of 5.5 hours. The automated microscope used is described in more detail in our co-pending application PCT/EP2018/085692.

MIC call

Images were analysed by converting them to called biomass values and MICs as described in WO 2017/216312. For different antibiotics, the microbial biomass at each time point was determined at different antibiotic concentrations (mg/l), such as Figure 14A (trimethoprim/sulfamethoxazole) and Figure 14B (piperacillin/tazol) Bataan).

result

MIC antibiotic concentrations (measured in mg/l) were determined for a range of antibiotics. In this experiment, each antibiotic was present in AST consumables in triplicate. The results are shown in Table 2.

Table 2 - MIC values

Claims (51)

1.A method of preparing a suspension of intact microorganisms from a sample comprising the microorganisms and mammalian cells, the method comprising:

a. providing a sample comprising a microorganism and mammalian cells;

b. contacting the sample with a buffer solution, a detergent, and one or more proteases, wherein the pH of the buffer solution is at least pH6 and less than pH 9, to lyse mammalian cells present in the sample;

c. filtering the mixture obtained in step (b) through a filter suitable for retaining microorganisms, wherein said filtering removes lysed mammalian cells from said mixture;

d. recovering the microorganisms retained by the filter in step (c), wherein the recovering comprises resuspending the microorganisms in a liquid to provide a suspension comprising recovered intact microorganisms; and

e. determining the concentration of microorganisms in the suspension, wherein the concentration of microorganisms is determined by a method comprising:

i. contacting an aliquot of the suspension with an alcohol and/or heating an aliquot of the suspension;

optionally diluting one or more aliquots of the suspension to provide one or more diluted aliquots at one or more dilution values, wherein the dilution is performed before, during and/or after step (i);

contacting at least a portion of the aliquot of step (e) (i) or (e) (ii) with a single fluorescent stain capable of binding DNA, wherein the stain has an emission wavelength, to provide a suspension-stain mixture;

imaging the suspension-stain mixture of step (e) (iii) at the emission wavelength of the fluorescent stain and determining an image analysis value for the number of objects corresponding to microorganisms in the imaged mixture; and

v. comparing the image analysis values obtained in step (e) (iv) of the aliquot of step (e) (iii) with a predetermined calibration curve, thereby determining the concentration of microorganisms in the suspension.

2. The method of claim 1, wherein the method further comprises a step (f) of adjusting the concentration of microorganisms in at least a portion of the suspension.

3. The method of claim 2, wherein step (f) comprises adjusting the concentration of microorganisms in at least a portion of the suspension prior to determining the concentration of microorganisms in the suspension.

4. A method according to claim 2 or 3, wherein step (f) comprises adjusting the concentration of the microorganisms in at least a portion of the suspension after the concentration is determined in step (e).

5. The method of any one of claims 2 to 4, wherein the concentration is adjusted by dilution.

6. The method of any one of claims 1 to 5, wherein the stain of step (e) (iii) is a cell permeable stain.

7. The method according to any one of claims 1 to 6, wherein the sample is a clinical or veterinary sample or a culture of a clinical or veterinary sample.

8. The method of any one of claims 1 to 7, wherein the buffer solution is at pH6.5 to 8.5, alternatively at pH6.5 to 8 or 7 to 8.

9. The method of any one of claims 1 to 8, wherein the buffer solution is at pH 7.5.

10. The method of any one of claims 1 to9, wherein the detergent is a non-ionic detergent.

11. The method of claim 10, wherein the non-ionic detergent is Brij-O10.

12. The method according to any one of claims 1 to11, wherein the concentration of the detergent is between 0.1 to 5% w/v, or between 0.1 to 1% w/v.

13. The method of claim 12, wherein the concentration of the detergent is 0.45% w/v.

14. The method according to any one of claims 1 to 13, wherein the protease is proteinase K.

15. The method of any one of claims 1 to 14, wherein step (b) comprises contacting the sample with a composition comprising: (i) a lysis buffer comprising PBS pH 7.5, 0.45% w/v Brij-O10, and (ii) proteinase K.

16. The method according to any one of claims 1 to 15, wherein the filtration step (c) comprises filtering the mixture using a filter having a pore size of less than 0.5 μ ι η, preferably less than 0.25 μ ι η.

17. The method of any one of claims 1-16, wherein recovering microorganisms from the filter comprises backwashing liquid through the filter.

18. The method according to any one of claims 1 to 17, wherein in step (d) the microbial cells are resuspended in a liquid growth medium suitable for culturing a microorganism.

19. The method of any one of claims 1 to 18, wherein the filter is washed between steps (c) and (d).

20. The process of any one of claims 1 to 19, wherein the alcohol of step (e) (i) is ethanol.

21. The process of claim 20, wherein in step (e) (i), ethanol is added to the suspension to a resulting concentration of 30 to 40% v/v, preferably 35% (v/v).

22. The method of any one of claims 1 to 21, wherein in step (e) (ii), the suspension is diluted with a buffer.

23. The method of claim 22, wherein the buffer is PBS.

24. The method of any one of claims 1 to 23, wherein the fluorescence intensity of the fluorescent stain at the emission wavelength increases when the stain binds to nucleic acids.

25. The method of claim 24, wherein the fluorescent stain is an asymmetric cyanine dye.

26. The method according to any one of claims 1 to 25, wherein the fluorescent stain has an excitation wavelength and an emission wavelength in the wavelength range of 350 and 700 nm.

27. The method of claim 26, wherein the fluorescent stain is a green fluorescent stain.

28. The method of claim 24 or 27, wherein the fluorescent stain is a SYTO stain.

29. The method of claim 28, wherein the SYTO stain is SYTO BC.

30. The method according to any one of claims 1 to 29, wherein the method comprises diluting an aliquot of the suspension to provide two or more diluted aliquots at different dilution values, wherein the two or more aliquots are prepared simultaneously, or sequentially during step (e) (ii), wherein a second or further diluted aliquot is prepared after steps (e) (iv) and/or (e) (v).

31. The method of claim 30, wherein steps (e) (iii) and (e) (iv) are performed on two or more aliquots at different dilution values, and wherein step (e) (v) comprises identifying an aliquot that includes an image analysis value that is within a predetermined calibration curve range, and comparing the image analysis value of the aliquot to the predetermined calibration curve, thereby determining the concentration of intact microorganisms in the suspension.

32. The method of claim 31, wherein steps (e) (iii) and (e) (iv) are performed simultaneously for each aliquot.

33. The method of claim 31, wherein steps (e) (iii) and (e) (iv) are performed sequentially for each aliquot.

34. The method of any one of claims 1 to 33, wherein images are obtained at one or more focal planes through the suspension-stain mixture.

35. The method of claim 34, wherein the imaging comprises obtaining a series of 2-D images along an optical axis, wherein each image is obtained at a different location along the optical axis through a volume of the suspension-stain mixture.

36. The method of any one of claims 1 to 35, wherein step (e) (iii) of contacting with the stain is performed at a temperature greater than 4 ℃.

37. The method of any one of claims 1 to 36, wherein in the contacting of step (e) (iii), the aliquot or diluted aliquot, or portion thereof, is incubated with the stain for a period of from 1 to 20 minutes.

38. The method of any one of claims 1 to 37, wherein the imaging in step (e) (iv) is performed at room temperature.

39. The method of any one of claims 1 to 38, wherein in the imaging of step (e) (iv), the microorganisms are identified as being aggregated or non-aggregated, and a predetermined calibration curve for aggregated or non-aggregated microorganisms is used.

40. The method of any one of claims 1 to 39, wherein the image is analyzed for fluorescence intensity and/or size of each enumerated object, and optionally morphology of each enumerated object.

41. The method of any one of claims 1 to 40, wherein the image is analyzed for maximum fluorescence intensity, median fluorescence intensity, and/or area of each enumerated object.

42. The method of any one of claims 1 to 41, wherein the image is analyzed for maximum, median and/or mean fluorescence intensity and/or area of population of objects.

43. A method for determining antimicrobial sensitivity of a microorganism in a sample, the method comprising:

(i) providing a sample comprising living microorganisms and mammalian cells;

(ii) subjecting the sample to steps (b) to (d) as defined in any one of claims 1 to 40 to produce a suspension of the living microorganisms;

(iii) performing step (e) as defined in any one of claims 1 to 40 to determine the concentration of microbial cells in the suspension;

(iv) (iii) inoculating a series of test microbial cultures for Antibiotic Susceptibility Testing (AST) using the suspension of step (ii), wherein the series of test microbial cultures comprises at least two different growth conditions, wherein the different growth conditions comprise one or more different antimicrobial agents, and each antimicrobial agent is tested at two or more different concentrations; and

(v) the degree of microbial growth under each growth condition was evaluated,

wherein the concentration of microbial cells in the suspension or the test microbial culture is adjusted to a desired or predetermined concentration, if necessary; and

wherein the extent of microbial growth under each growth condition is used to determine at least one value indicative of the sensitivity of a microorganism in the sample to at least one antimicrobial agent.

44. The method of claim 43, wherein at least one MIC and/or SIR value is determined to determine antimicrobial sensitivity of the microorganism in the sample.

45. The method of claim 43 or claim 44, wherein the concentration of at least a portion of the suspension of step (ii) is adjusted based on the concentration determined in step (iii) to provide an inoculum for inoculating the test microbial culture in step (iv).

46. The method of any one of claims 43 to 45, wherein the step of adjusting the concentration comprises dilution based on the concentration determined in step (iii).

47. A method according to claim 46 wherein after step (iii), at least a portion of the suspension of step (ii) is diluted to provide the inoculum for step (iv).

48. The method of any one of claims 43 to 47, wherein the concentration of microorganisms in the inoculated microbial test culture is at 4.5x10⁵80% or 5x10⁵In the range of ± 60%.

49. The method of any one of claims 43-48, wherein at least one of the test microbial cultures comprises a fastidious medium.

50. The method of any one of claims 43-45 or 48-49, wherein concentration adjusting comprises culturing or further culturing the suspension.

51. The method of any one of claims 43 to 50, wherein if the concentration of microorganisms in the suspension is below 1x10⁶And (c) microorganisms, the AST assay is not performed on the suspension.

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